Next Article in Journal
Multivariate Genome-Wide Association Study of Concentrations of Seven Elements in Seeds Reveals Four New Loci in Russian Wheat Lines
Previous Article in Journal
Response of Aboveground Net Primary Production, Species and Phylogenetic Diversity to Warming and Increased Precipitation in an Alpine Meadow
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:

A Comprehensive Review Uncovering the Challenges and Advancements in the In Vitro Propagation of Eucalyptus Plantations

School of Bioengineering and Bioscience, Lovely Professional University, Phagwara 144411, Punjab, India
DBS (PG) College, Dehradun 248001, Uttarakhand, India
Himalayan School of Biosciences, Swami Rama Himalayan University, Jolly Grant Dehradun 248016, Uttarakhand, India
Department of Bioresource and Food Science, Institute of Natural Science and Agriculture, Konkuk University, Seoul 05029, Republic of Korea
Author to whom correspondence should be addressed.
Plants 2023, 12(17), 3018;
Submission received: 27 June 2023 / Revised: 3 August 2023 / Accepted: 14 August 2023 / Published: 22 August 2023
(This article belongs to the Special Issue Plant Tissue Culture IV)


The genus Eucalyptus is a globally captivated source of hardwood and is well known for its medicinal uses. The hybrid and wild species of Eucalyptus are widely used as exotic plantations due to their renowned potential of adapting to various systems and sites, and rapid large-scale propagation of genetically similar plantlets, which further leads to the extensive propagation of this species. Tissue culture plays a crucial role in the preservation, propagation, and genetic improvement of Eucalyptus species. Despite unquestionable progression in biotechnological and tissue culture approaches, the productivity of plantations is still limited, often due to the low efficiency of clonal propagation from cuttings. The obtained F1 hybrids yield high biomass and high-quality low-cost raw material for large-scale production; however, the development of hybrid, clonal multiplication, proliferation, and post-developmental studies are still major concerns. This riveting review describes the problems concerning the in vitro and clonal propagation of Eucalyptus plantation and recent advances in biotechnological and tissue culture practices for massive and rapid micropropagation of Eucalyptus, and it highlights the Eucalyptus germplasm preservation techniques.

1. Introduction

Eucalyptus (Family: Myrtaceae) is a genus including rapidly growing evergreen ornamental trees and shrubs indigenous to Australia. Over 900 different species native to Australia, Argentina, Brazil, Chile, France, India, Indonesia, Portugal, Morocco, Spain, South Africa, and the USA have been planted, covering more than 20 million hectares of land [1,2]. The augmented demand for its pulp and hardwood as well as its adaptivity to various biotic stressors and high productivity have hiked its economic value [3]. Besides these, the essential oils concentrated in different organs of this plant possess biological, toxicological, and pharmacological applications, thus having a promising prospect for ethnomedicine [4]. Owing to the increased future demands, rapidly propagating Eucalyptus plantations are desired [5,6].

1.1. Challenges with Conventional Breeding

The conventional breeding programs involve the cultivation of Eucalyptus from seeds; however, challenges such as long generation time and genetic load persist [7]. These challenges have paved the way for modern biotechnological and plant tissue culture approaches, allowing for highly efficient rapid multiplication of plants in in vitro environments as well as germplasm storage and preservation [8]. Biotechnological tools, along with traditional tree improvement techniques, can be considered as solutions for meeting the growing human demand for forest products as compared to conventional tree improvement programs alone; also, biotechnology opens doors for understanding a wide range of complex biological problems related to forest tree species. Forest biotechnology covers a wide array of modern techniques covering aspects of genetic engineering, in vitro regeneration techniques, genomics, metabolomics, proteomics, molecular markers, and marker-assisted breeding. These advanced biotechnological tools have opened new doors for understanding the genetic structure, identifying biotic and abiotic interactions and stress tolerance factors, and linking some of the important genetic traits, thus facilitating accelerated selection and further breeding programs. One such efficient technique is micropropagation. The promising approach of micropropagation is achieved via organogenesis and somatic embryogenesis [9]. The history of Eucalyptus tissue culture can be traced back to the mid-20th century, and it has since become a significant tool in the conservation, propagation, and breeding of Eucalyptus species. Early experiments in the 1950s and 1960s faced challenges due to the limited knowledge of plant growth regulators and maintaining sterile conditions. However, the 1970s and 1980s marked a significant breakthrough, when pioneering researchers in Australia, such as Dr. Carron and Dr. Griffin, successfully developed protocols for the mass propagation of Eucalyptus species using tissue culture techniques. This success led to practical applications in the forestry industry, where tissue culture provided disease-free and genetically identical Eucalyptus plantlets for reforestation and commercial plantations. As techniques advanced, tissue culture became essential in Eucalyptus genetic improvement and breeding programs, focusing on enhancing desired traits such as growth rate and disease resistance. Cryopreservation techniques were also introduced to effectively conserve Eucalyptus germplasm. Despite challenges like somaclonal variation and disease outbreaks, ongoing research aims to improve tissue culture protocols for Eucalyptus species. Eucalyptus tissue culture continues to play a vital role in sustainable forestry practices, biodiversity conservation, and genetic preservation of valuable Eucalyptus species.
This paper attempts to discuss the methods of clonal propagation in upcoming sections. This study also focuses on the factors that influence the clonal propagation of Eucalyptus and further discusses the advances in germplasm preservation techniques. Previous research concerning traditional and modern breeding programs for improving Eucalyptus plantations in the last 25 years is included.

1.2. A Crop with Potential Biomass Production

As reported, nine dominating species of Eucalyptus have been cultivated for hardwood, namely E. camaldulensis, E. pellita, E. dunnii, E. grandis, E. tereticornis, E. globulus, E. sargila, E. uropylla, and E. nitens [10]. The F1 hybrids achieved from the breeding of these species show seed vigor and yield high biomass. Moreover, the F1 hybrids from interspecific breeding show heterosis. Successful attempts to achieve commercial-scale plantations have been made for some species (E. deglupta, E. grandis, E. camaldulensis, and E. pellita) agile to tropical and subtropical regions with high rainfall via macro- and microcuttings [10,11,12]. For those species unable to rapidly propagate using these methods, tissue culture is a propitious approach [13]. Events of natural hybridization between Eucalyptus species have been reported [14]. The spontaneous hybrid formation between E. benthamii and E. dunnii at Embrapa forests in Colombo, Paraná State, presented frost-tolerant varieties. Likewise, artificially pollinated hybrids serve as a viable alternative for future forest plantations while combining the useful traits from different parental combinations [15,16]. Initially, thirteen mature F1 Eucalyptus hybrid pairings were reported in India. Studies were initiated to create controlled and natural hybrids using half-sibling progeny generated from the seeds gathered from the stands of two intercross able species growing close to one another based on cross-ability patterns. The hybrids were generated mechanically or naturally through selection in the Dehradun campus of the Forest Research Institute’s New Forest area. Researchers had worked on two important interspecific F1 hybrids, namely FRI-5 (E. camaldulensis Dehn × E. tereticornis Sm) and FRI-14 (E. torelliana F.V. Muell × E. citriodora Hook), among several interspecific hybrids that were created [17]. The superiority of Eucalyptus hybrids FRI-5 and FRI-14 to their parentage has been established. Additionally, high levels of hybrid vigor have been observed in these plants, and they outperformed the parent combinations by three to five times in terms of growth characteristics. The average yield per unit area per unit of time was lower in the F2 population produced from seeds because of the significant segregation observed [17].
According to [18,19], these hybrids produced more biomass than their parents’ offspring and the Mysore Gum. The authors of these studies reported a micropropagation strategy for cloning and large-scale production as novel clonal propagation techniques for the commercial cultivation of these priceless Eucalyptus hybrids. The hybrid was observed to be intermediate to its parental species in more than half of the contrasting characteristics tested; this is noteworthy because of the two parent species involved, i.e., E. grandis and E. tereticornis. The former has a faster growth rate and good stem form, provides the best pulp quality, and prefers areas with high rainfall, while the latter is drought-tolerant, showing the suitability of hybrids toward intermediary environments (hybrid habitat). The outcomes of this hybrid micropropagation were reported for the first time in India. The species and hybrids that are reproduced via traditional methods (such as the many millions of E. grandis × E. urophylla and E. grandis plants produced per year) are often adapted to heavy rainfall areas in the tropics or subtropics. On drier, colder sites where land is more easily accessible and less expensive, hardwood plantations are being planted more frequently. Other Eucalyptus species are needed for these locations, such as species that are harder to propagate from cuttings, including C. citriodora, E. globulus, E. cloeziana F. Muell., or E. nitens [20,21]. The development of effective techniques for the clonal propagation of Eucalyptus, particularly for species that are difficult to propagate from cuttings, has remained a major issue in hardwood forestry. As tissue culture is among the most promising methods to rapidly propagate desirable genotypes and maintain germplasm in vitro, it has been used sustainably for these species and many others by various researchers [22].

2. Micropropagation and Its Applications

Achieving true-to-type plants with desirable traits using in vitro protocols is termed micropropagation (Figure 1 and Figure 2). Five critical stages need to be accomplished to successfully establish micropropagation [23]. These five stages include (i) Stage 0, defined as the preparatory stage for developing efficient and reproducible protocol; (ii) Stage 1, which aims to establish an aseptic and viable culture; (iii) Stage 2, which involves propagation without immolating objective to be achieved; (iv) Stage 3, involving large-scale propagation; and (v) Stage 4, the hardening stage of the established plant material. Each of the stages involved is discussed comprehensively in subsequent sections. Figure 3 illustrates different events of Eucalyptus micropropagation.

2.1. Establishing Axenic Culture

For ensuring the success of micropropagation, maintaining uncontaminated cultures are necessary throughout the protocol. The foremost important step in the in vitro propagation of plants is to establish a microbial contamination-free culture because the primary explants employed are nonsterile and thus a major source of microbial contamination in the culture [24]. The nodal segments bearing axillary buds, shoot tips, lead discs, and seeds could be used as explants to initiate tissue culture. The use of seeds as explants does not lead to true-to-type propagation; however, due to the ease of decontamination and juvenility of young seedlings, this method is superior for rapid clonal propagation [25,26]. Moreover, dormant axillary buds from nodes are widely used for maintaining clonal fidelity, providing thousands of plantlets via rapid propagation with high multiplication rates. The propagation from nodal segments is also reported in the micropropagation of woody plants like bamboo [27]. Nonetheless, the explants from leaf discs and shoot tips provide true-to-type propagation, although they are least preowned due to onerous sterilization [25,27]. The initial step involves the surface sterilization of the explant, which is usually achieved by first rinsing/washing the explant with nonsterilized water, immersing it in ethyl alcohol (70%), or treating it with chlorine-based sterilant such as calcium hypochlorite (Ca(OCl)2, mercuric chloride (HgCl2), and sodium hypochlorite (NaOCl), followed by rinsing with sterile distilled water. To prevent drying and increase the interaction between the explant surface and sterilant, a drop of wetting agents such as Tween 20 is often added. Although chlorine-based sterilants can efficiently sterilize the explants, their use is associated with toxic effects on living cells. Mercuric chloride has been reported to cause mammalian toxicity. So, the use of either calcium hypochlorite or sodium hypochlorite is recommended [28,29]. The basic explant sterilization of Eucalyptus is achieved by treating it with sodium hypochlorite at 67–1340 mM concentrations. However, the concentrations of sodium hypochlorite must be optimized before experimentation as the concentration affects the overall success of micropropagation. For instance, as reported, the seed germination of Corymbia citridora and Corymbia torelliana hybrid was decreased when the concentration of NaOCl was increased from 134 to 670 mM [30]. The optimization of the sterilization protocol of five different clones of eucalypt was performed using 1% sodium hypochlorite, 0.1% mercuric chloride, 1 mg/mL of rifampicin, and 70% ethanol as sterilant; the study suggested that, along with 1% sodium hypochlorite and 70% ethanol, using 0.1% mercuric chloride for 3 min was optimum, and using 1 mg/mL of rifampicin for 5 min was optimum for effective sterilization [31].
Leaching out of phenolics from cultured explants is also a main concern that prevents the establishment of in vitro cultures to a large extent. Reports suggest that nodal segments cultured on MS medium exudated phenolics. The number of phenolics exudated by nodal segments and bud-break response varied with the month of the collection of explants. Nodal segments were reported to be collected every month from January to December. It was found that explants collected during April and July to September showed the least phenolic exudation and better bud-break response comparatively and were best for in vitro studies. The phenolic exudation was high from October to January and May to June with poor bud-break responses [32].

2.2. In Vitro Proliferation of Shoot

In plant tissue culture, two main pathways are involved in achieving shoot regeneration: embryogenesis and organogenesis. Both processes play crucial roles in the development of new shoots, but they differ in the cellular mechanisms and morphological changes they undergo. The previous literature has only focused on “shoot multiplication”; however, for a better understanding, we provide a concise explanation for organogenesis and embryogenesis in the next sections.

2.2.1. Organogenesis

Organogenesis refers to the process through which shoot regeneration occurs via the differentiation and development of new shoots from pre-existing meristematic cells or tissues. In this pathway, the initial step involves the induction of adventitious shoots from explants such as leaf, stem, or root segments. The explants are cultured on a nutrient medium supplemented with specific plant growth regulators, particularly cytokinin, which stimulate the formation of shoot primordia. These primordia then undergo further growth and differentiation to develop into shoots with organized structures, including leaves and stems. Organogenesis is often characterized by the initiation of multiple shoots, referred to as shoot proliferation or multiplication [33]. The formation of new organs directly from explants is termed direct organogenesis, while the formation of new organs from cell cultures or suspensions, tissues, or calluses is termed indirect organogenesis [33,34,35]. Furthermore, organogenesis also includes the regeneration of roots from explants, which is known as direct root organogenesis, wherein new roots are directly developed from explants. The development of new organs depends on a variety of factors, such as hormones and type of media [36]. The differential media and nutrients employed in Eucalyptus micropropagation are discussed in the next section. Mostly basal MS salts and Murashige Skoog media of different strengths are used for establishing organogenesis in Eucalyptus. Besides these, Woody Plant Medium; Driver and Kuniyaki Walnut medium; Juan, Antonio, Diva, and Salvin medium; Schenk and Hildebrandt medium; and B5 medium have also been extensively used [17,33,37].

2.2.2. Somatic Embryogenesis

The development of bipolar embryos from somatic cells/tissues asexually is termed somatic embryogenesis [38]. It is a pathway for shoot, root, and plantlet regeneration that mimics the process of embryo development in plants that involves the dedifferentiation of somatic cells to a totipotent state, where they regain the ability to form an embryonic structure. Somatic embryos can be formed via two pathways, direct embryogenesis in which the embryo develops from pre-embryonic cells, or indirect embryogenesis in which the embryo develops from a callus grown on culture media. Pre-embryonic cells are undifferentiated cells that have the potency to differentiate into any kind of cells. Generally, apical meristems, hypocotyls, and epicotyls serve as a source of pre-embryonic cells as these contain undifferentiated cells [33,38]. A protocol for somatic embryogenesis has been reported by [39], suggesting that the leaves from adult trees are better for inducing somatic embryogenesis than floral tissues. The dedifferentiated cells progress through various stages of embryo development, including the formation of a proembryo, globular embryo, heart-shaped embryo, and mature embryo. Shoots arise from these embryogenic structures and can be further multiplied through subsequent subculturing. However, the inability of somatic embryos to reach the maturation stage is an adverse limitation of clonal propagation, the success of which is directly dependent upon the optimization of PGRs used, the age of tissue used, and the type of media used for establishment (Table 1). Typically, semi-solid MS media supplemented with sucrose are used to initiate Eucalyptus somatic embryogenesis; however, B5 media supplemented with sucrose have also been reported to induce somatic embryogenesis in C. citridora. Moreover, various PGRs have also been reported to induce embryogenesis in Eucalyptus; for instance, a hormone-free medium for inducing somatic embryogenesis was also suggested [40,41,42,43].

2.3. Adventitious Root Formation and Root Hardening

The bipolar structure formed after somatic embryogenesis can directly germinate by using nutrients from basal media for the shoot and root proliferation. Contrastingly, the unipolar structure needs the proliferation of adventitious roots at the base of their shoots for the development of plantlets. This is usually achieved via semi-solid media; often, activated charcoal is also introduced in these media as it regulates the pH of the media and is also reported to adsorb the inhibitory compounds from the media, in addition to reducing irradiance at the base of the shoots [57,58,59]. Improved in vitro rooting in E. grandis × E. nitens was reported by reducing the strength of MS media from full to half-strength and decreasing the concentration of sucrose in shooting media from 20 to 15 g L−1. Additionally, it was observed that increasing the concentration of NAA from 0.1 mg L−1 to 0.5 mg L−1 increased the average percentage of adventitious roots [29]. Also, increasing the IBA concentration from 0.1 mg L−1 to 0.5 mg L−1 increased root hair formation. Similar studies on E. erythronema × E. stricklandii suggested 8 weeks of continuous exposure to IBA on roots resulted in the longest root length [44]. Light studies on E. grandis × E. urophylla suggested the use of red–blue light to be superior for rooting and showed the highest mean number of roots [56,60].
Before adventitious root formation, the acclimatization of shoots is necessary for their future success in nursery conditions. Micropropagated plantlets were hardened using a liquid MS medium (1/4 strength) with 2% sucrose. Furthermore, for supporting roots, adsorbent cotton was soaked in this liquid medium. After maintaining for 2 weeks, the plantlets were transferred to mist bags containing a 1:1:1 ratio of soil, manure, and sand and then transferred to a net house. Finally, the plantlets were transferred to field conditions and showed 85–95% success rates in field conditions [17]. However, in another study, 58% of the success rate of plantlets in field conditions was due to the loss of some plantlets during handling [29]. Another innovative approach for acclimatization was reported, where the shoots were maintained in photoautotrophic culture at a high concentration of carbon dioxide and a low sugar concentration. These conditions promote carbon fixation and transpiration. Notably, 86–96% of success rate for E. camaldulensis and 100% success rate for E. grandis × E. urophylla have been achieved with this method [45,61,62,63]. A similar study suggested that increasing the temperature from 18–13 °C to 33–28 °C increased the number of root cuttings per stock plant [46]. Moreover, improved rooting efficiency was achieved in clones of E. urophylla via in vitro rejuvenation/reinvigoration [64].

3. Factors Affecting the Efficiency of Micropropagation

The efficiency of micropropagation can be influenced by various factors. Growth regulators play a crucial role in promoting shoot proliferation and rooting in tissue culture. The type and concentration of cytokinins and auxins in the culture medium can significantly impact the rate and quality of shoot formation. Additionally, the composition of the culture medium, including the types and concentrations of nutrients, vitamins, and carbon sources, affects the growth and development of explants. Organic elements such as amino acids and complex organic compounds can enhance shoot regeneration and plantlet growth, while inorganic elements, including macronutrients and micronutrients, are vital for overall plant health and development. Moreover, light is an essential environmental factor that affects micropropagation efficiency. Proper light intensity and photoperiod can influence shoot initiation, elongation, and rooting. The spectrum of light, particularly the red-to-far-red light ratio, also plays a role in regulating plant growth and differentiation. Optimizing these factors in micropropagation protocols is crucial for maximizing the efficiency of shoot proliferation and the production of healthy plantlets. Here, we discuss all these factors and their effects on Eucalyptus micropropagation.

3.1. Role of Plant Growth Regulators

Plant growth regulators or plant growth hormones are chemical compounds widely recognized to alter the growth of plants, for instance, to suppress the growth of shoots, boost the growth of shoots, or alter the maturity of the fruit. Indole-3-acetic acid (IAA) is a natural auxin in plants, and indole-3-butyric acid (IBA) is the analog of the auxin found in plants. Both IAA and IBA are synthesized via tryptophan-dependent or tryptophan-independent pathways [65,66]. Both IAA and IBA can be quickly metabolized in tissues of Eucalyptus plants. Since auxins play a crucial role in the regulation of cell division, as well as the elongation of plants and many other phases of their development, these are stored by plants as either IAA or IBA, which is converted into IAA whenever required [67,68].
Plant growth regulators have been reported to, directly and indirectly, influence plantlet proliferation, including the differentiation of embryos and different organs. For instance, optimal concentrations of cytokinin–auxin necessary for inducing organogenesis in E. cloeziana micropropagation were suggested by [47]. Moreover, the highest rate of somatic embryogenesis was also reported by introducing 0.1 mg L−1 NAA and 0.5 mg L−1 BA [48]. Increased shoot multiplication via the addition of 0.5 mg L−1 BAP in WPM and ½ MS medium was also reported [49]. It is important to note that the optimization of PGRs in culture media is necessary as they could negatively affect the growth of plantlets [49,69]. Similar studies on auxin types in E. salgina and E. globulus were reported, suggesting the best rooting obtained with IBA than IAA. Best rooting was achieved in both species when treated with IBA; the possible explanation supporting this IAA is that it is highly susceptible to enzymatic degradation and is also 5 times more susceptible to photo-oxidation than IBA. In support of this, a similar study on E. sideroxylon micorcuttings was reported by comparing IBA and NAA using different concentrations of both auxins from 0 to 10 μM. A high frequency of callus induction was reported by culturing cotyledon explants on MS media supplemented with 1 mg L−1 NAA + 0.5 mg L−1 BA. The same study also suggested that MS media supplemented with 0.5 mg L−1 NAA + 1 mg L−1 BA + 1 mg L−1 GA3, as well as ½ MS media supplemented with 0.5–1 mg L−1, resulted in high-frequency adventitious root formation in E. bosistoana. Furthermore, 100% survival of preacclimatized plantlets was obtained [70]. A similar study revealed best shoot elongation by supplementing the media with 0.05 mg L−1 BAP + 1 mg L−1 NAA and 0.05 mg L−1 BAP + 1 mg L−1 NAA + 1 mg L−1 IBA−1 [71]. The results showed increased callus induction in micorcuttings exposed to IBA compared with the micorcuttings exposed to NAA. However, the responses to auxins may vary from species to species based on their differential affinities, uptake, and metabolization of auxins [72,73].

3.2. Effect of Culture Media

The initiation of shoot proliferation is usually achieved by culturing explant on a semi-solid medium that comprises gelling agents such as 1.5–4.0 g/L of gelrite, 4–8 g/L of agar, or 1.5–4.5 g/L of phytagel, and the pH is adjusted between 5 and 6. Also, the use of liquid media has been reported for the initiation and proliferation of nodes and shoots [74,75]. The culture of the shoot depends on the ability to encourage the development of axillary and accessory buds that are present at the base of each leaf axil. In previous research attempts at Eucalyptus micropropagation, the basal media used include Murashige and Skoog media (different strengths ½, ¼); JADS (Juan, Antonio, Diva, and Silvian) medium, WPM (Woody Plant Medium), and DKW (Driver and Kuniyaki Walnut) medium [76]. Additionally, the form of medium, whether a liquid suspension medium or a semi-solid medium, affects the growth of plantlets in vitro. Reportedly, better shoot multiplication of Eucalyptus has been observed in liquid media than in semi-solid media [77]. WPM was reported as the optimal medium for the micropropagation of E. benthamii [50]. Similarly, the JADS medium was observed to be optimal for the trunk base shoot elongation of E. grandis. The DKW medium was used as an alternative for the micropropagation of E. nitens [51]. The adjustment of the pH of the medium to 5.8 prior to autoclaving at 121 °C for 15 min has been recommended. Temperature incubation at 25 ± 2 °C and 16 h photoperiod with the photon flux density of 2500 lux from white fluorescent tubes is the recommended cultured condition for Eucalyptus spp. For improving the survival of the explant, polyvinyl pyrrolidone, activated charcoal, and ascorbate have also been added to culture media. However, the type of media and the response of plantlets vary among species of Eucalyptus and their hybrids. Moreover, some drawbacks such as chlorosis, tissue browning, and oxidation have been reported in almost all types of media used for Eucalyptus micropropagation.

3.3. Importance of Organic and Inorganic Elements

Organic and inorganic elements play a crucial role in plant micropropagation media, which are used for the propagation and growth of plants under sterile conditions. Elements like calcium (Ca), nitrogen (N), phosphorous (P), and boron (B) serve as macro- and micronutrients essential for nourishing plant growth. These have been introduced in in vitro cultures to promote the proliferation and differentiation of organs from the shoot. An appropriate balance and concentration of these organic and inorganic elements are crucial to ensure the successful propagation and growth of plants in vitro [78]. The composition of the medium can be adjusted based on the specific requirements of different plant species and their growth stages. The elements required by plants in concentrations lower than 0.5 mM/L are referred to as micronutrients, and the elements with more than this concentration are referred to as macronutrients [79,80,81]. Magnesium (Mg), calcium (Ca), hydrogen (H), sulfur (S), potassium (K), nitrogen (N), phosphorous (P), and oxygen (O) serve as macronutrients. Calcium act as an important cofactor and cellular messenger involved in various signal transduction pathways and is well known to play various important roles in plant stress [82]. For instance, in E. urophylla and E. grandis, calcium has been reported to trigger organogenesis [83]. Manganese (Mn), chlorine (Cl), iron (Fe), zinc (Zn), boron (B), sodium (Na), iodine (I), and copper (Cu) serve as the microelements among which iron is the most critical element. Also, it was reported that the deficiency of boron in media led to necrosis and callus accumulation, further inhibiting seedling growth in E. grandis [84]. Furthermore, a study suggested that calcium, in the form of calcium chloride in agitated liquid media, decreases the hyperhydricity in E. saligna. However, due to the toxicity caused by chlorine, it had been not effective in completely eliminating hyperhydricity [85]. Calcium chloride dihydrate was also reported to induce shoot elongation and decrease vitrification [86]. Nonetheless, efforts in improving the optimization and choice of organic/inorganic elements have been increased, although they are insufficient in considering the individual role of vast available macro- and micronutrients.

3.4. The Role of Carbohydrates

Carbohydrates are important biomolecules that provide biofuel and serve as a carbon source for cell growth. Different reducing and nonreducing sugars are available and have been employed in micropropagation like glucose, fructose, sucrose, and galactose, among which sucrose is still the most preferable in Eucalyptus micropropagation due to its ease of translocation in plant tissues. Sucrose is a nonreducing sugar, specifically a disaccharide composed of fructose and glucose. Some of the reports suggest that increased sucrose concentrations can hinder water and nutrient uptake in plants and inhibit photosynthesis by influencing photosynthetic enzymes. However, contrastingly, some studies suggest that plants remain uninfluenced by high sucrose concentrations. Furthermore, in E. cloiziana, high glucose and sucrose concentrations were reported to decrease the shoot length (conc. > 15 g/mL in media) [87]. For instance, studies reported different concentrations of sucrose (1–6%) in MS media for in vitro shoot proliferation. The best results were reported using 3% sucrose in MS media with a 6–7-fold increased shoot multiplication. Similar findings reported that media devoid of sucrose result in the inhibition of shoot multiplication, and the leaves and shoots turned to a pale green color [88,89]. The results of this study are in line with those of several studies that used 3% sucrose as a carbohydrate source to promote the growth of shoots in a variety of Eucalyptus species. However, similar findings on many other woody plants have also been reported; for instance, in bamboo shoots, successful multiplication was observed when media were supplemented with 2% sucrose [90,91]. It has also been reported that an increase in sucrose levels from 3 to 4% does not cause any effect on shoots but results in albinism. Similarly, at 1% sucrose concentration, thin shoots and leaves were developed that were inappropriate for subculturing. An investigation was conducted on myo-inositol to determine its role in in vitro shoot multiplication. MS media supplemented with 100 mg L−1 of myo-inositol yielded the best shoot multiplication rates, while MS media devoid of myo-inositol showed decreased shoot multiplication. Moreover, MS media supplemented with excess myo-inositol (more than 150 mg L−1) not only decreased shoot multiplication but also had detrimental effects on shoots. For maximizing shoot multiplication rate and growth, 100 mg L−1 myo-inositol was supplemented in culture media for all trials [6,88].

3.5. Effects of Radiation and Light Exposure

Light is a critical external aspect influencing the different phases of plant growth. Light hour durations and intensity are directly linked to plants’ photosynthetic rates [92]. Many hybrids have been studied that suggest the effects of light and radiation on the success of micropropagation. The effects of five sources of lights, namely fluorescent lamps, white LEDs, red LEDs, blue LEDs, and red–blue LEDs, on E. grandis × E. urophylla hybrid were studied, and red–blue LEDs and florescent lights were found to be superior for E. grandis × E. urophylla micropropagation. The response of Eucalyptus to micropropagation varies among genotypes. Moreover, a low level of irradiation triggered rooting in E. globulus; contrastingly, some studies confirmed that low-level irradiations hindered root proliferation in E. globulus [52,53,60]. Also, studies on E. salgina and E. globulus were conducted for the effect of light on rooting capacity using white fluorescent lamps. E. globulus did not show any effect from exposure and was found to be dependent only on exogenous auxin concentration for rooting, while E. salgina cuttings showed increased root density per rooted cutting upon exposure to light combined with exogenous auxin application [93]. A similar study suggested that preservation under low light intensity effectively preserved cultures for 3 months [54]. Besides these, increases in light intensity and carbon dioxide content have been shown to increase the growth of explants photoautotrophically [94]. In a similar study, the effect of light quality on the clone of E. urophylla in the photoautotrophic system was assessed, focusing on the stomatal density, carotenoid content, chlorophyll content, the number of shoots, and the longest shoot. The results indicated that blue LED resulted in fewer shoots, while high production of carotenoids was observed under white light [95]. In another study on Eucalyptus dunnii and Eucalyptus grandis × E. urophylla, it was observed that the use of white light was associated with increased buds per plant, decreased tissue oxidation, and longer shoot length. In E. dunnii, blue, red, and yellow light resulted in increased chlorophyll a and b content. Also, blue, white, purple, and red light increased stomatal densities. Moreover, a previous study revealed that irrespective of light spectra, E. dunnii showed decreased adventitious rooting [96]. Another similar study on E. grandis × E. urophylla clone was carried out to assess the impact of five different light sources, namely fluorescent lamps as well as blue, green, red, and yellow cellophane light in a bioreactor system. Yellow and blue light sources were found to be more suitable for the clone as less hyperhydricity was observed along with spongy parenchymatic tissue, thicker mesophyll, increased shoot length, and more shoots per explant [97]. Moreover, another study suggests that for the in vitro multiplication of E. pilularis, white light was more suitable, and for the E. urograndis clone, blue light was more suitable because it increased the number of buds, shoots length, and fresh weight per explant [98]. Table 1 and Table 2 highlights critical factors in Eucalyptus micropropagation.

4. In Vitro Germplasm Preservation

In vitro germplasm preservation plays a vital role in conserving plant biodiversity, protecting endangered species, and safeguarding important genetic resources for future research, breeding programs, and restoration efforts. It helps to maintain a diverse and resilient gene pool, ensuring the availability of plant materials for sustainable agriculture, forestry, and environmental conservation. Brief insights into recent preservation techniques that are cryopreservation and cold storage in Eucalyptus spp. are discussed below.
The preservation of germplasm is peremptory for breeding programs. Cryopreservation is a protocol that involves germplasm storage at ultra-lower temperatures (−135 °C to −196 °C) in liquid nitrogen. At this temperature, the cell viability and genetic stability are preserved; however, the aging of the cell is hindered due to the halting of all biochemical and physiological pathways of the cell [99]. Various reports on successful cryopreservation of Eucalyptus spp. have been reported, including shoot tips of E. grandis × E. camaldulensis, E. urophylla × E. grandis, E. grandis, E. grandis × E. urophylla, and E. camaldulensis using the droplet vitrification method, firstly by preculturing shoot tips on MS media containing 0.25 M sucrose concentration for 24 h and then again on MS media containing 0.625 M sucrose concentration for 24 h, followed by subjecting the individual shoot tips to plant vitrification solution (PVS). Similarly, the preservation of the axillary buds of E. grandis × E. camaldulensis has also been reported by placing the axillary buds in semi-solid MS media and significantly increasing the concentration of sucrose and glycerol from 0.4 to 0.7 to 1.0 M; this type of method showed 49% of regrowth [55,99,100].
Cold storage involves the preservation of plants at lower temperatures. Some attempts have been made to preserve shoots of E. grandis in cold storage using half- and full-strength media. However, half-strength MS media allowed for the preservation of shoots at 24 °C to 28 °C for 10 months; contrastingly full-strength media could preserve the same shoots at 10 °C only for 6 months. Similar studies were conducted on Corymbia toleriana and Corymbia citridora and showed the preservation of shoots on full-strength MS media at 14 °C for 12 months [101].

5. Limitations, Challenges, and Future Directions

Even though successful attempts in micropropagation have been made, some challenges like hyperhydricity, phenolic oxidation, explant contamination, and root and ex vitro survival of the established clone still persist. Addressing these problems requires more effort and refinement of tissue culture protocols, including the optimization of growth media, hormone formulations, and culture conditions. Strict aseptic techniques and effective sterilization methods are crucial to minimizing endogenous contamination. Moreover, future directions in exploring novel approaches such as genetic engineering for improving the performance of propagated clones and utilizing “muti-omics” approaches for understanding the molecular basis of various stress, responses, and key regulators affecting the overall efficiency of propagated plants can help in enhancing the success of micropropagation. This comprehensive review holistically addressed the gaps in the large-scale propagation of the Eucalyptus plants, thus helping the scientific community with further research in a useful direction. Overcoming these challenges will contribute to the successful large-scale propagation of forest species through in vitro techniques, which will benefit reforestation efforts and conservation initiatives.

6. Conclusions

Micropropagation has allowed for achieving rapid clonal plantations; however, the efficiency and success of micropropagation depends on the rate of shoot multiplication. Eucalyptus spp. and its hybrids are among the commercially important tree spps and have been extensively propagated via micropropagation. These are continuously studied for their genetic improvement and establishment of superior hybrid species. The micropropagation of Eucalyptus can be achieved in four main steps: (i) suitable explant collection, (ii) the preparation of aseptic culture, (iii) shoot and root proliferation, and (iv) the hardening of the root. A wide range of media, both semi-solid and liquid culture media, have been described for the efficient clonal propagation of Eucalyptus. Currently, many studies are still being conducted for the development of improved tissue culture protocols as well as for improving the wood quality of Eucalyptus. For achieving success in the callogenesis and histogenesis of Eucalyptus spp., complete knowledge of all optimization procedures, including the type of media, as well as the choice of hormones, their concentrations, and ratios, must be known. Apart from that, the age and type of tissues, as well as the seasons during which explants are collected, are necessary to consider for ensuring the success of tissue proliferation. Moreover, the field success of plantlets is necessary, for which proper conditions and protocols for the acclimatization of plantlets must be known. All of these important factors have been compiled in the present study. However, more studies are required to close the gap between the in vitro development of plantlets of Eucalyptus and their growth in field conditions, which is important for its industrial success.

Author Contributions

Conceptualization, V.S. and I.S.; methodology, A.K. and V.S.J.; writing—original draft preparation, V.S., A., A.K. and B.K.; writing—review and editing, S.S., A.K. and V.S.J.; visualization, V.S.J. and S.G.; supervision, V.S. and I.S.; project administration, I.S.; funding acquisition, I.S. All authors have read and agreed to the published version of the manuscript.


This research received no external funding.

Data Availability Statement

Data are contained within this article.


This article was supported by the KU Research Professor Program of Konkuk University.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Watt, M.P.; Blakeway, F.C.; Mokotedi, M.E.O.; Jain, S.M. Micropropagation of Eucalyptus. In Micropropagation of Woody Trees and Fruits; Jain, S.M., Ishii, K., Eds.; Springer Netherlands: Dordrecht, The Netherlands, 2003; Volume 75, pp. 217–244. [Google Scholar] [CrossRef]
  2. Trueman, S.J.; Hung, C.D.; Wendling, I. Tissue Culture of Corymbia and Eucalyptus. Forests 2018, 9, 84. [Google Scholar] [CrossRef]
  3. Nogueira, M.C.D.J.A.; Araujo, V.A.D.; Vasconcelos, J.S.; Christoforo, A.L.; Lahr, F.A.R. Sixteen Properties of Eucalyptus tereticornis Wood for Structural Uses. Biosci. J. 2020, 36, 449–457. [Google Scholar] [CrossRef]
  4. Silva, J.; Abebe, W.; Sousa, S.M.; Duarte, V.G.; Machado, M.I.L.; Matos, F.J.A. Analgesic and Anti-Inflammatory Effects of Essential Oils of Eucalyptus. J. Ethnopharmacol. 2003, 89, 277–283. [Google Scholar] [CrossRef]
  5. Kamal, B.; Arya, I.; Sharma, V.; Jadon, V.S. In Vitro Enhanced Multiplication and Molecular Validation of Eucalyptus F1 Hybrids. Plant Cell Biotechnol. Mol. Biol. 2016, 17, 167–175. [Google Scholar]
  6. Arya, I.D.; Chauhan, S.S.S.; Arya, S. Micropropagation of Superior Eucalyptus Hybrids FRI-5 (Eucalyptus camaldulensis Dehn × E. tereticornis Sm) and FRI-14(Eucalyptus Torelliana F.V. Muell × E. citriodora Hook): A Commercial Multiplication and Field Evaluation. Afr. J. Biotechnol. 2009, 8, 5718–5726. [Google Scholar] [CrossRef]
  7. Kendurkar, S.V.; Rangaswamy, M. Genetic Transformation in Eucalyptus. In Biotechnologies of Crop Improvement, Volume 2; Gosal, S.S., Wani, S.H., Eds.; Springer International Publishing: Cham, Switzerland, 2018; pp. 335–366. [Google Scholar] [CrossRef]
  8. El-Esawi, M.A. Micropropagation Technology and Its Applications for Crop Improvement. In Plant Tissue Culture: Propagation, Conservation and Crop Improvement; Anis, M., Ahmad, N., Eds.; Springer: Singapore, 2016; pp. 523–545. [Google Scholar] [CrossRef]
  9. Trueman, S.J. Clonal Propagation and Storage of Subtropical Pines in Queensland, Australia. S. Afr. For. J. 2006, 208, 49–52. [Google Scholar] [CrossRef]
  10. Booth, T.H. Eucalypt Plantations and Climate Change. Ecol. Manag. 2013, 301, 28–34. [Google Scholar] [CrossRef]
  11. Vilasboa, J.; Da Costa, C.T.; Fett-Neto, A.G. Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root Eucalyptus spp. Plants 2022, 11, 3281. [Google Scholar] [CrossRef]
  12. Shanthi, K.; Bachpai, V.K.W.; Anisha, S.; Ganesan, M.; Anithaa, R.G.; Subashini, V.; Chakravarthi, M.; Sivakumar, V.; Yasodha, R. Micropropagation of Eucalyptus camaldulensis for the Production of Rejuvenated Stock Plants for Microcuttings Propagation and Genetic Fidelity Assessment. New For. 2015, 46, 357–371. [Google Scholar] [CrossRef]
  13. Kataria, V.; Masih, A.; Chauhan, S.; Sharma, S.K.; Kant, A.; Arya, I.D. Clonal Propagation, a Tested Technique for Increasing Productivity: A Review of Bamboos, Eucalyptus and Chirpine. In Agricultural Biotechnology: Latest Research and Trends; Kumar Srivastava, D., Kumar Thakur, A., Kumar, P., Eds.; Springer Nature: Singapore, 2021; pp. 37–51. [Google Scholar] [CrossRef]
  14. Butcher, P.A.; Skinner, A.K.; Gardiner, C.A. Increased Inbreeding and Inter-Species Gene Flow in Remnant Populations of the Rare Eucalyptus benthamii. Conserv. Genet. 2005, 6, 213–226. [Google Scholar] [CrossRef]
  15. Potts, B.M.; Dungey, H.S. Interspecific Hybridization of Eucalyptus: Key Issues for Breeders and Geneticists. New For. 2004, 27, 115–138. [Google Scholar] [CrossRef]
  16. Jovanovic, T.; Arnold, R.; Booth, T. Determining the Climatic Suitability of Eucalyptus dunnii for Plantations in Australia, China and Central and South America. New For. 2000, 19, 215–226. [Google Scholar] [CrossRef]
  17. Singh, D.; Kaur, S.; Kumar, A. In Vitro Drought Tolerance in Selected Elite Clones of Eucalyptus tereticornis Sm. Acta Physiol. Plant 2020, 42, 17. [Google Scholar] [CrossRef]
  18. Venkatesh, C.S.; Sharma, V.K. Hybrid Vigour in Controlled Interspecific Crosses of Eucalyptus tereticornis × E. camaldulensis. Silvae Genet. 1977, 26, 121–124. [Google Scholar]
  19. Paramathma, M.; Surendran, C.; Rai, R.S.V. Studies on heterosis in six Eucalyptus species. J. Trop. For. Sci. 1997, 9, 283–293. [Google Scholar]
  20. De Almeida, M.R.; De Bastiani, D.; Gaeta, M.L.; De Araujo Mariath, J.E.; De Costa, F.; Retallick, J.; Nolan, L.; Tai, H.H.; Strömvik, M.V.; Fett-Neto, A.G. Comparative Transcriptional Analysis Provides New Insights into the Molecular Basis of Adventitious Rooting Recalcitrance in Eucalyptus. Plant Sci. 2015, 239, 155–165. [Google Scholar] [CrossRef] [PubMed]
  21. Dickinson, G.R.; Wallace, H.M.; Lee, D.J. Reciprocal and Advanced Generation Hybrids between Corymbia citriodora and C. torelliana: Forestry Breeding and the Risk of Gene Flow. Ann. For. Sci. 2013, 70, 1–10. [Google Scholar] [CrossRef]
  22. Wendling, I.; Warburton, P.; Trueman, S. Maturation in Corymbia torelliana × C. citriodora Stock Plants: Effects of Pruning Height on Shoot Production, Adventitious Rooting Capacity, Stem Anatomy, and Auxin and Abscisic Acid Concentrations. Forests 2015, 6, 3763–3778. [Google Scholar] [CrossRef]
  23. Debergh, P.C.; Read, P.E. Micropropagation. In Micropropagation: Technology and Application; Debergh, P.C., Zimmerman, R.H., Eds.; Springer Netherlands: Dordrecht, The Netherlands, 1991; pp. 1–13. [Google Scholar] [CrossRef]
  24. Cassells, A.C. Pathogen and Biological Contamination Management in Plant Tissue Culture: Phytopathogens, Vitro Pathogens, and Vitro Pests. In Plant Cell Culture Protocols; Loyola-Vargas, V.M., Ochoa-Alejo, N., Eds.; Humana Press: Totowa, NJ, USA, 2012; Volume 877, pp. 57–80. [Google Scholar] [CrossRef]
  25. Wendling, I.; Brooks, P.R.; Trueman, S.J. Topophysis in Corymbia torelliana × C. citriodora Seedlings: Adventitious Rooting Capacity, Stem Anatomy, and Auxin and Abscisic Acid Concentrations. New For. 2015, 46, 107–120. [Google Scholar] [CrossRef]
  26. Bag, N.; Chandra, S.; Palni, L.M.S.; Nandi, S.K. Micropropagation of Dev-Ringal [Thamnocalamus spathiflorus (Trin.) Munro]—A Temperate Bamboo, and Comparison between in Vitro Propagated Plants and Seedlings. Plant Sci. 2000, 156, 125–135. [Google Scholar] [CrossRef]
  27. Giri, C.C.; Shyamkumar, B.; Anjaneyulu, C. Progress in Tissue Culture, Genetic Transformation and Applications of Biotechnology to Trees: An Overview. Trees 2004, 18, 115–135. [Google Scholar] [CrossRef]
  28. Ansar, S.; Iqbal, M. Effect of Dietary Antioxidant on Mercuric Chloride Induced Lung Toxicity and Oxidative Stress. Toxin Rev. 2015, 34, 168–172. [Google Scholar] [CrossRef]
  29. Keret, R.; Nakhooda, M.; Jones, N.B.; Hills, P.N. Optimisation of Micropropagation Protocols for Temperate Eucalypt Hybrids in South Africa, with a Focus on Auxin Transport Proteins. South. For. J. For. Sci. 2021, 83, 254–263. [Google Scholar] [CrossRef]
  30. Trueman, S.J.; Richardson, D.M. In Vitro Propagation of Corymbia torelliana × C. citriodora (Myrtaceae) via Cytokinin-Free Node Culture. Aust. J. Bot. 2007, 55, 471. [Google Scholar] [CrossRef]
  31. Kuppusamy, S.; Ramanathan, S.; Sengodagounder, S.; Senniappan, C.; Shanmuganathan, R.; Brindhadevi, K.; Kaliannan, T. Optimizing the Sterilization Methods for Initiation of the Five Different Clones of the Eucalyptus Hybrid Species. Biocatalaysis Agric. Biotechnol. 2019, 22, 101361. [Google Scholar] [CrossRef]
  32. Kamal, B.; Arya, I.D.; Gupta, S. In-Vitro Regeneration of Interspecific F1 Hybrid (Eucalyptus citriodora and Eucalyptus torelliana) of Eucalyptus. J. Mt. Res. 2022, 17, 125–130. [Google Scholar] [CrossRef]
  33. Aggarwal, D.; Kumar, A.; Reddy, M.S. Shoot Organogenesis in Elite Clones of Eucalyptus tereticornis. Plant Cell Tiss. Organ. Cult 2010, 102, 45–52. [Google Scholar] [CrossRef]
  34. Girijashankar, V. In Vitro Regeneration of Eucalyptus camaldulensis. Physiol. Mol. Biol. Plants 2012, 18, 79–87. [Google Scholar] [CrossRef]
  35. Fernando, S.C.; Goodger, J.Q.D.; Gutierrez, S.S.; Johnson, A.A.T.; Woodrow, I.E. Plant Regeneration through Indirect Organogenesis and Genetic Transformation of Eucalyptus polybractea R.T. Baker. Ind. Crops Prod. 2016, 86, 73–78. [Google Scholar] [CrossRef]
  36. Sluis, A.; Hake, S. Organogenesis in Plants: Initiation and Elaboration of Leaves. Trends Genet. 2015, 31, 300–306. [Google Scholar] [CrossRef]
  37. Oberschelp, G.P.J.; Gonçalves, A.N. Assessing the Effects of Basal Media on the in Vitro Propagation and Nutritional Status of Eucalyptus dunnii Maiden. In Vitro Cell. Dev. Biol.—Plant 2016, 52, 28–37. [Google Scholar] [CrossRef]
  38. Méndez-Hernández, H.A.; Ledezma-Rodríguez, M.; Avilez-Montalvo, R.N.; Juárez-Gómez, Y.L.; Skeete, A.; Avilez-Montalvo, J.; De-La-Peña, C.; Loyola-Vargas, V.M. Signaling Overview of Plant Somatic Embryogenesis. Front. Plant Sci. 2019, 10, 77. [Google Scholar] [CrossRef]
  39. Martínez, M.T.; del San-José, M.C.; Arrillaga, I.; Cano, V.; Morcillo, M.; Cernadas, M.J.; Corredoira, E. Holm Oak Somatic Embryogenesis: Current Status and Future Perspectives. Front Plant Sci 2019, 10, 239. [Google Scholar] [CrossRef]
  40. Pinto, G.; Silva, S.; Park, Y.-S.; Neves, L.; Araújo, C.; Santos, C. Factors Influencing Somatic Embryogenesis Induction in Eucalyptus globulus Labill.: Basal Medium and Anti-Browning Agents. Plant Cell Tiss. Organ Cult. 2008, 95, 79–88. [Google Scholar] [CrossRef]
  41. Pinto, G.; Park, Y.-S.; Neves, L.; Araújo, C.; Santos, C. Genetic Control of Somatic Embryogenesis Induction in Eucalyptus globulus Labill. Plant Cell Rep. 2008, 27, 1093–1101. [Google Scholar] [CrossRef]
  42. Pinto, G.; Park, Y.-S.; Silva, S.; Neves, L.; Araújo, C.; Santos, C. Factors Affecting Maintenance, Proliferation, and Germination of Secondary Somatic Embryos of Eucalyptus globulus Labill. Plant Cell Tiss. Organ. Cult. 2008, 95, 69–78. [Google Scholar] [CrossRef]
  43. Pinto, G.; Silva, S.; Neves, L.; Araújo, C.; Santos, C. Histocytological Changes and Reserve Accumulation during Somatic Embryogenesis in Eucalyptus globulus. Trees 2010, 24, 763–769. [Google Scholar] [CrossRef]
  44. Glocke, P.; Delaporte, K.; Collins, G.; Sedgley, M. Micropropagation of Juvenile Tissue of Eucalyptus erythronema × Eucalyptus stricklandii Cv. ‘Urrbrae Gem’. In Vitro Cell. Dev. Biol.—Plant 2006, 42, 139–143. [Google Scholar] [CrossRef]
  45. Tanaka, M.; Giang, D.T.T.; Murakami, A. Application of a Novel Disposable Film Culture System to Photoautotrophic Micropropagation of Eucalyptus uro-grandis (Urophylia × grandis). In Vitro Cell. Dev. Biol.—Plant 2005, 41, 173–180. [Google Scholar] [CrossRef]
  46. Trueman, S.J.; McMahon, T.V.; Bristow, M. Production of Eucalyptus cloeziana Cuttings in Response to Stock Plant Temperature. J. Trop. For. Sci. 2013, 25, 60–69. [Google Scholar]
  47. De Oliveira, L.S.; Brondani, G.E.; Molinari, L.V.; Dias, R.Z.; Teixeira, G.L.; Gonçalves, A.N.; De Almeida, M. Optimal Cytokinin/Auxin Balance for Indirect Shoot Organogenesis of Eucalyptus cloeziana and Production of Ex Vitro Rooted Micro-Cuttings. J. For. Res. 2022, 33, 1573–1584. [Google Scholar] [CrossRef]
  48. Prakash, M.G.; Gurumurthi, K. Effects of Type of Explant and Age, Plant Growth Regulators and Medium Strength on Somatic Embryogenesis and Plant Regeneration in Eucalyptus camaldulensis. Plant Cell Tiss. Organ Cult. 2010, 100, 13–20. [Google Scholar] [CrossRef]
  49. Brondani, G.E.; Dutra, L.F.; Wendling, I.; Grossi, F.; Hansel, F.A.; Araujo, M.A. Micropropagation of an Eucalyptus hybrid (Eucalyptus benthamii × Eucalyptus dunnii). Acta Sci. Agron. 2011, 33, 8317. [Google Scholar] [CrossRef]
  50. Brondani, G.E.; de Wit Ondas, H.W.; Baccarin, F.J.B.; Gonçalves, A.N.; de Almeida, M. Micropropagation of Eucalyptus benthamii to Form a Clonal Micro-Garden. In Vitro Cell. Dev. Biol.—Plant 2012, 48, 478–487. [Google Scholar] [CrossRef]
  51. Gomes, F.; Canhoto, J.M. Micropropagation of Eucalyptus nitens Maiden (Shining Gum). In Vitro Cell. Dev. Biol.—Plant 2003, 39, 316–321. [Google Scholar] [CrossRef]
  52. Mankessi, F.; Saya, A.; Baptiste, C.; Nourissier, S.; Monteuuis, O. In Vitro Rooting of Genetically Related Eucalyptus urophylla × Eucalyptus grandis Clones in Relation to the Time Spent in Culture. Trees 2009, 23, 931–940. [Google Scholar] [CrossRef]
  53. Fett-Neto, A.G.; Fett, J.P.; Goulart, L.W.V.; Pasquali, G.; Termignoni, R.R.; Ferreira, A.G. Distinct Effects of Auxin and Light on Adventitious Root Development in Eucalyptus saligna and Eucalyptus globulus. Tree Physiol. 2001, 21, 457–464. [Google Scholar] [CrossRef] [PubMed]
  54. Watt, M.P.; Thokoane, N.L.; Mycock, D.; Blakeway, F. In Vitro Storage of Eucalyptus grandis Germplasm under Minimal Growth Conditions. Plant Cell Tissue Organ Cult. 2000, 61, 161–164. [Google Scholar] [CrossRef]
  55. Blakesley, D.; Kiernan, R. Cryopreservation of Axillary Buds of a Eucalyptus grandis × Eucalyptus camaldulensis Hybrid. Cryo Lett. 2001, 22, 13–18. [Google Scholar]
  56. Souza, D.M.S.C.; Fernandes, S.B.; Avelar, M.L.M.; Frade, S.R.D.P.; Molinari, L.V.; Gonçalves, D.S.; Pinto, J.E.B.P.; Brondani, G.E. Light Quality in Micropropagation of Eucalyptus grandis × Eucalyptus urophylla. Sci. For. 2020, 48, e3329. [Google Scholar] [CrossRef]
  57. Thomas, T.D. The Role of Activated Charcoal in Plant Tissue Culture. Biotechnol. Adv. 2008, 26, 618–631. [Google Scholar] [CrossRef]
  58. Jones, N.B.; van Staden, J. Micropropagation and Establishment of Eucalyptus grandis Hybrids. S. Afr. J. Bot. 1994, 60, 122–126. [Google Scholar] [CrossRef]
  59. Sapaeing, A.; Sutthinon, P.; Hilae, A.; Wattanapan, N. Effects of BA, NAA, and Activated Charcoal on Micropropagation of Nepenthes Mirabilis (Lour.) Druce. Acta Hortic. 2020, 1298, 281–286. [Google Scholar] [CrossRef]
  60. Xu, Y.; Liang, Y.; Yang, M. Effects of Composite LED Light on Root Growth and Antioxidant Capacity of Cunninghamia lanceolata Tissue Culture Seedlings. Sci. Rep. 2019, 9, 9766. [Google Scholar] [CrossRef] [PubMed]
  61. Murthy, H.N.; Joseph, K.S.; Paek, K.Y.; Park, S.Y. Bioreactor Systems for Micropropagation of Plants: Present Scenario and Future Prospects. Front. Plant Sci. 2023, 14, 1159588. [Google Scholar] [CrossRef]
  62. Zobayed, S. Mass Propagation of Eucalyptus camaldulensis in a Scaled-up Vessel Under In Vitro Photoautotrophic Condition. Ann. Bot. 2000, 85, 587–592. [Google Scholar] [CrossRef]
  63. Kozai, T.; Afreen, F.; Zobayed, S.M.A. (Eds.) . Photoautotrophic (Sugar-Free Medium) Micropropagation as a New Micropropagation and Transplant Production System; Springer: Berlin/Heidelberg, Germany, 2005. [Google Scholar] [CrossRef]
  64. Mendonça, E.G.; Batista, T.R.; Stein, V.C.; Balieiro, F.P.; de Abreu, J.R.; Pires, M.F.; de Souza, P.A.; Paiva, L.V. In Vitro Serial Subculture to Improve Rooting of Eucalyptus urophylla. New For. 2020, 51, 801–816. [Google Scholar] [CrossRef]
  65. Leva, A. (Ed.) Recent Advances in Plant In Vitro Culture; InTech: London, UK, 2012. [Google Scholar] [CrossRef]
  66. Zhao, Y. Auxin Biosynthesis. Arab. Book 2014, 12, e0173. [Google Scholar] [CrossRef]
  67. Frick, E.M.; Strader, L.C. Roles for IBA-Derived Auxin in Plant Development. J. Exp. Bot. 2018, 69, 169–177. [Google Scholar] [CrossRef] [PubMed]
  68. Strader, L.C.; Bartel, B. Transport and Metabolism of the Endogenous Auxin Precursor Indole-3-Butyric Acid. Mol. Plant. 2011, 4, 477–486. [Google Scholar] [CrossRef] [PubMed]
  69. Nazirah, A.; Nor-Hasnida, H.; Mohd-Saifuldullah, A.W.; Muhammad-Fuad, Y.; Ahmad-Zuhaidi, Y.; Rozidah, K. Development of an Efficient Micropropagation Protocol for Eucalyptus Hybrid (E. urophylla × E. grandis) through axillary shoot proliferation. J. Trop. For. Sci. 2021, 33, 391–397. [Google Scholar] [CrossRef]
  70. Shwe, S.S.; Leung, D.W.M. Plant Regeneration from Eucalyptus bosistoana Callus Culture. In Vitro Cell. Dev.Biol.—Plant 2020, 56, 718–725. [Google Scholar] [CrossRef]
  71. Faria, J.C.T.; Ribeiro-Kumara, C.; Costa, R.S.D.R.; Nieri, E.M.; De Carvalho, D.; Pinto, J.E.B.P.; Neto, A.R.D.S.; Brondani, G.E. Use of Biodegradable Polyester-Based Microvessels for Micropropagation of Mature Eucalyptus microcorys. N. Z. J. For. Sci. 2022, 52, 1–13. [Google Scholar] [CrossRef]
  72. Skůpa, P.; Opatrný, Z.; Petrášek, J. Auxin Biology: Applications and the Mechanisms Behind. In Applied Plant Cell Biology; Nick, P., Opatrny, Z., Eds.; Springer: Berlin/Heidelberg, Germany, 2014; Volume 22, pp. 69–102. [Google Scholar] [CrossRef]
  73. Teale, W.D.; Paponov, I.A.; Palme, K. Auxin in Action: Signalling, Transport and the Control of Plant Growth and Development. Nat. Rev. Mol. Cell. Biol. 2006, 7, 847–859. [Google Scholar] [CrossRef]
  74. Bunn, E. Development of in Vitro Methods for Ex Situ Conservation of Eucalyptus impensa, an Endangered Mallee from Southwest Western Australia. Plant Cell. Tiss. Organ. Cult. 2005, 83, 97–102. [Google Scholar] [CrossRef]
  75. Kaur, S. In Vitro Regeneration of Shoots From Nodal Explants of Dendrobium chrysotoxum Lindl. J. Hortic. Res. 2017, 25, 27–34. [Google Scholar] [CrossRef]
  76. Woodward, A.W.; Bartel, B. Auxin: Regulation, Action, and Interaction. Ann. Bot. 2005, 95, 707–735. [Google Scholar] [CrossRef]
  77. Chen, J.; Ziv, M. The Effect of Ancymidol on Hyperhydricity, Regeneration, Starch and Antioxidant Enzymatic Activities in Liquid-Cultured Narcissus. Plant Cell Rep. 2001, 20, 22–27. [Google Scholar] [CrossRef]
  78. Ngomuo, M.; Mneney, E.; Ndakidemi, P.A. The In Vitro Propagation Techniques for Producing Banana Using Shoot Tip Cultures. Am. J. Plant Sci. 2014, 05, 1614–1622. [Google Scholar] [CrossRef]
  79. Al-Aizari, A.A.; Al-Obeed, R.S.; Mohamed, M.A.H. Improving Micropropagation of Some Grape Cultivars via Boron, Calcium and Phosphate. Electron. J. Biotechnol. 2020, 48, 95–100. [Google Scholar] [CrossRef]
  80. George, E.F.; Hall, M.A.; Klerk, G.-J.D. (Eds.) The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients. In Plant Propagation by Tissue Culture; Springer Netherlands: Dordrecht, The Netherlands, 2007; pp. 65–113. [Google Scholar] [CrossRef]
  81. Pérez-Tornero, O.; Burgos, L. Different Media Requirements for Micropropagation of Apricot Cultivars. Plant Cell Tissue Organ Cult. 2000, 63, 133–141. [Google Scholar] [CrossRef]
  82. White, P.J.; Broadley, M.R. Calcium in Plants. Ann. Bot. 2003, 92, 487–511. [Google Scholar] [CrossRef] [PubMed]
  83. Al-Mayahi, A.M.W. Effect of Calcium and Boron on Growth and Development of Callus and Shoot Regeneration of Date Palm ‘Barhee’. Can. J. Plant Sci. 2020, 100, 357–364. [Google Scholar] [CrossRef]
  84. Brunoni, F.; Rolli, E.; Dramis, L.; Incerti, M.; Abarca, D.; Pizarro, A.; Diaz-Sala, C.; Ricci, A. Adventitious Rooting Adjuvant Activity of 1,3-Di(Benzo[d]Oxazol-5-Yl)Urea and 1,3-Di(Benzo[d]Oxazol-6-Yl)Urea: New Insights and Perspectives. Plant Cell Tiss. Organ Cult. 2014, 118, 111–124. [Google Scholar] [CrossRef]
  85. Lopes da Silva, A.L.; Gollo, A.; Brondani, G.; Horbach, M.; Oliveira, L.; Machado, M.; Lima, K.; Costa, J. Micropropagation of Eucalyptus saligna Sm. from Cotyledonary Nodes. Pak. J. Bot. 2015, 47, 311–318. [Google Scholar]
  86. Sharma, S.; Ramamurthy, V. Micropropagation of 4-Year-Old Elite Eucalyptus tereticornis Trees. Plant Cell Rep. 2000, 19, 511–518. [Google Scholar] [CrossRef] [PubMed]
  87. Gago, D.; Vilavert, S.; Bernal, M.Á.; Sánchez, C.; Aldrey, A.; Vidal, N. The Effect of Sucrose Supplementation on the Micropropagation of Salix viminalis L. Shoots in Semi-solid Medium and Temporary Immersion Bioreactors. Forests 2021, 12, 1408. [Google Scholar] [CrossRef]
  88. Joshi, I.; Bisht, P.; Sharma, V.K.; Uniyal, D.P. In Vitro Clonal Propagation of Mature Eucalyptus F1 Hybrid (Eucalyptus tereticornis Sm. x E. grandis Hill Ex. Maiden). Silvae Genet. 2003, 52, 110–113. [Google Scholar]
  89. Gago, D.; Bernal, M.Á.; Sánchez, C.; Aldrey, A.; Cuenca, B.; Christie, C.B.; Vidal, N. Effect of Sucrose on Growth and Stress Status of Castanea sativa × C. crenata Shoots Cultured in Liquid Medium. Plants 2022, 11, 965. [Google Scholar] [CrossRef]
  90. Sandhu, M.; Wani, S.H.; Jiménez, V.M. In Vitro Propagation of Bamboo Species through Axillary Shoot Proliferation: A Review. Plant Cell Tiss Organ Cult. 2018, 132, 27–53. [Google Scholar] [CrossRef]
  91. Nadgauda, R.S.; Parasharami, V.A.; Mascarenhas, A.F. Precocious Flowering and Seeding Behaviour in Tissue-Cultured Bamboos. Nature 1990, 344, 335–336. [Google Scholar] [CrossRef]
  92. Shen, G.; Tan, S.; Sun, X.; Chen, Y.; Li, B. Experimental Evidence for the Importance of Light on Understory Grass Communities in a Subtropical Forest. Front Plant Sci. 2020, 11, 1051. [Google Scholar] [CrossRef] [PubMed]
  93. Fogaça, C.M.; Fett-Neto, A.G. Role of Auxin and Its Modulators in the Adventitious Rooting of Eucalyptus Species Differing in Recalcitrance. Plant Growth Regul. 2005, 45, 1–10. [Google Scholar] [CrossRef]
  94. Xiao, Y.; Niu, G.; Kozai, T. Development and Application of Photoautotrophic Micropropagation Plant System. Plant Cell Tiss. Organ Cult. 2011, 105, 149–158. [Google Scholar] [CrossRef]
  95. Miranda, N.A.; Xavier, A.; Otoni, W.C.; Gallo, R.; Gatti, K.C.; de Moura, L.C.; Souza, D.M.S.C.; Maggioni, J.H.; de Santos, S.S.O. Quality and Intensity of Light in the In Vitro Development of Microstumps of Eucalyptus urophylla in a Photoautotrophic System. For. Sci. 2020, 66, 754–760. [Google Scholar] [CrossRef]
  96. Rangel Do Prado Frade, S.; Santana Costa Souza, D.M.; Fernandes, S.B.; Lopes Martins Avelar, M.; Vaz Molinari, L.; Santos Gonçalves, D.; Alves Magalhães, T.; Brondani, G.E. Spectral Quality Influence on in Vitro Morphophysiological Responses of Eucalyptus dunnii Maiden and Eucalyptus grandis W.Hill Ex Maiden × E. urophylla ST Blake. N. Z. J. For. Sci. 2023, 53, 1–16. [Google Scholar] [CrossRef]
  97. Souza, D.M.S.C.; Avelar, M.L.M.; Fernandes, S.B.; Silva, E.O.; Duarte, V.P.; Molinari, L.V.; Brondani, G.E. Spectral Quality and Temporary Immersion Bioreactor for in Vitro Multiplication of Eucalytpus grandis × Eucalyptus urophylla. 3 Biotech 2020, 10, 457. [Google Scholar] [CrossRef]
  98. Matheus, D.; Souza, S.C.; Martins, A.R.; Fernandes, S.B.; Lopes Martins Avelar, M.; Vaz Molinari, L.; Santos Gonçalves, D.; Brondani, G.E. In Vitro Multiplication of Eucalyptus pilularis and Eucalyptus grandis × E. urophylla (Urograndis Eucalypt): Effect of Light Quality in Temporary Immersion Bioreactor. Mindanao J. Sci. Technol. 2022, 20, 72–86. [Google Scholar]
  99. Roque-Borda, C.A.; Kulus, D.; Vacaro De Souza, A.; Kaviani, B.; Vicente, E.F. Cryopreservation of Agronomic Plant Germplasm Using Vitrification-Based Methods: An Overview of Selected Case Studies. Int. J. Mol. Sci. 2021, 22, 6157. [Google Scholar] [CrossRef]
  100. Padayachee, K.; Watt, M.; Edwards, N.; Mycock, D. Cryopreservation as a Tool for the Conservation of Eucalyptus Genetic Variability: Concepts and Challenges. South. For. 2009, 71, 165–170. [Google Scholar] [CrossRef]
  101. Hung, C.D.; Trueman, S.J. Preservation of Encapsulated Shoot Tips and Nodes of the Tropical Hardwoods Corymbia torelliana × C. citriodora and Khaya Senegalensis. Plant Cell Tiss. Organ Cult. 2012, 109, 341–352. [Google Scholar] [CrossRef]
Figure 1. Schematic illustration of Eucalyptus micropropagation presenting sources of explant and basic steps involved in the protocol.
Figure 1. Schematic illustration of Eucalyptus micropropagation presenting sources of explant and basic steps involved in the protocol.
Plants 12 03018 g001
Figure 2. The steps involved in Eucalyptus micropropagation start with the collection and preparation of explants, which could be an axial leaf, shoot tips, buds, or seeds, followed by inoculating and culturing in specific media with the optimized concentration of the hormones and other nutritional supplements based on the requirement of the plant as well as environmental conditions. After shoot proliferation, the developed plants are subjected to root proliferation and root hardening in field conditions, or the germplasm of the developed plants could be preserved for future use in similar related protocols.
Figure 2. The steps involved in Eucalyptus micropropagation start with the collection and preparation of explants, which could be an axial leaf, shoot tips, buds, or seeds, followed by inoculating and culturing in specific media with the optimized concentration of the hormones and other nutritional supplements based on the requirement of the plant as well as environmental conditions. After shoot proliferation, the developed plants are subjected to root proliferation and root hardening in field conditions, or the germplasm of the developed plants could be preserved for future use in similar related protocols.
Plants 12 03018 g002
Figure 3. (AF) Different stages in Eucalyptus micropropagation: (A) aseptic shoot/explant inoculation; (B) aseptic culture establishment; (C) shoot proliferation; (D) in vitro rhizogenesis of microshoots; (E) hardening; (F) acclimatization and field transfer.
Figure 3. (AF) Different stages in Eucalyptus micropropagation: (A) aseptic shoot/explant inoculation; (B) aseptic culture establishment; (C) shoot proliferation; (D) in vitro rhizogenesis of microshoots; (E) hardening; (F) acclimatization and field transfer.
Plants 12 03018 g003
Table 1. The table below highlights various sources of explants, culture media, additives, etc., that have been previously used in Eucalyptus micropropagation.
Table 1. The table below highlights various sources of explants, culture media, additives, etc., that have been previously used in Eucalyptus micropropagation.
Explant-Source/TypeCulture Media/PGRs/AdditivesExperimental Outcomes/Remarks/Productivity/Root Hardening, etc.References
Nodal segmentsMS media, WPM, 0.5 mg L−1 IBA, 1.0 mg L−1 BAP, B5, NAA, KinetinFull-strength MS media supplemented with 1.0 mg L−1 BAP showed the best shoot elongation; ½ MS media supplemented with 0.5 mg L−1 IBA showed the best rooting; the resulting hybrid produced yielded 3–5 times more wood than parental species.[5]
Nodal segments (30–32-year trees)MS media, WPM, B5 medium, SH medium, IBA, BAP, NAA,Best in vitro studies were obtained from the explants collected during the period of January to February and August to September; ½ MS media + BAP resulted in best rooting (92%); up to 98% of plants survived acclimatization.[6]
Stem cuttings and axillary budsMS Media, 2.22 µm BA, 1.16 µm Kinetin, 0.029 µm gibberellic acid, 400 mg L−1 PVP, 30 g L−1 sucroseMicropropagation and microcuttings showed higher adventitious rooting (24.8–100% and 43–95%, respectively) than stem cuttings (9.3–75.5%).[12]
Maintained Elite clones (KE8, CE2, T1, and Y8)Basal MS media, 2.5 µM BA, 0.5 µM NAA, D-mannitol (0, 250, 500, 750, and 1000 mM)The culture growth index of all clones was reduced significantly because of drought stress.[17]
Cuttings from plants grown in vitro Full and ½ strength MS mediaConcentrations of IAA, IBA, and stem anatomy had no effect on the rooting potential of shoots.[22]
Nodal cuttingsIAA, IBA (0, 1, 3, 8 g Kg−1)The position from which the explant is harvested can affect the rooting potential and seed vigor. Explants obtained from 7/8 and 9/10 apical positions showed enhanced rooting and shooting.[25]
Cuttings from 6-month-old parental plantsMS media, IAA, meta-topolin, kinetin, BAP, vit. B5, biotin, sucrose0.5 mg L−1 meta-topolin and 1 mg L−1 IAA enhanced shoot elongation as well as bud proliferation, while 0.5 mg L−1 IAA resulted in the most consistent rooting percentages. Moreover, equal expression of AUX1 and PIN1 transporter genes increased responsiveness toward PGRs.[29]
Nodal segmentsMS media, 58 mM sucrose, 0.5 μM NAA, 2.5 μM BA,Media supplemented with 1.0 μM 2,4-D, 5.0 μM BA and 500 mg L−1 cefotaxime showed maximum (44.6%) shoot bud organogenesis.[33]
Nodal segmentsMS media, 2 mg L−1 BAP, 0.1 mg L−1 NAAMedia supplemented with 0.5 mg L−1 showed the best shoot elongation, ½ MS + 1 mg L−1 IAA showed the best root induction and elongation, and direct regeneration was observed in MS + 20:1 BAP: NAA.[34]
Young shoot segmentsWPM, MS media, 2iP, NAA, BAP, sucroseMedia supplemented with BA resulted in 99% shoot proliferation, media supplemented with 2iP resulted in 93% shoot regeneration, and IBA promoted rooting in 60% of the clones.[35]
Nodal segmentsEDM basal media (a novel basal media for E. dunnii) supplemented with 20 g L−1 sucrose and without PGRsHigher Fe, Cu, Zn, and Mn concentrations in EDMm media increased rooting. Moreover, high S and K concentrations in EDMm increased growth rate and multiplication. Also, no Fe chlorosis/oxidation was observed in shoots cultured on EDMm.[37]
Zygotic embryoOne of the following media and growth regulators: ½ or full-strength MS media/WPM/B5/DKW/JADS media/3 mg L−1 NAA/10 mL−1 silver nitrate/0.5 mg L−1 DTT/100 mg L−1 ascorbic acid/0.5 mg L−1 DTE/1% m/v PVP/1% m/v PVPP/0.01% w/v activated charcoalThe best media for somatic embryogenesis were B5 and MS. Moreover, Silver nitrate, activated charcoal, and DTE reduced the browning of explants.[41]
Somatic embryosMS media supplemented with 3 mg L−1 NAAMS medium without PGRs is highly efficient for promoting cotyledonary embryo proliferation and germination.[42]
Zygotic embryo cotyledonsHormone-free MS mediaThe reserve accumulation of mature zygotic embryos was analyzed. Cotyledonary somatic embryos possess a low density of starch and no lipids/proteins.[43]
Axillary shoots½ MS, 4.4 μM, 1 μM NAA, 1 g L−1 sucroseWPM and QL media supplemented with Gibberellic acids showed enhanced shoot proliferation, ½ WPM supplemented with 20 μM IBA showed enhanced rooting, and 67% Plantlet hardening was achieved.[44]
Shoot segmentsMS media, 0.02 mg L−1 IBAVitron vessel placed in Low Photon Flux density at 3000 ppm CO2 for 24 h/day yielded the best growth and quality of plantlets.[45]
Seedlings grown from seedsVariable potting mixtureLow temperatures of 18 °C/13 °C to 23 °C/18 °C (day/night) reduced the number of harvested cuttings; however, they did not affect the percentage of roots proliferated from cuttings. By contrast, increasing the temperature to 33 °C/28 °C resulted in an increased number of cutting per stock plant.[46]
Hypocotyl segments and cotyledonary leavesMS media supplemented with different concentrations of NAA and TDZ, 0.8 g L−1 PVP, 0.1 g L−1 biotin, 0.1 g L−1 calcium pantothenate, 30 g L−1 sucrose0.44 µM BAP increased the regeneration of adventitious buds.[47]
Zygotic embryos and cotyledonsMS media supplemented with 3 g L−1 sucrose and different concentrations of NAA, 2,4-D, BA, ABA1 mg L−1 NAA resulted in maximum callus induction, the frequency of callus proliferation depends on the age of the explant, with 10-year-old explants showing maximum proliferation, the highest frequency of somatic embryogenesis was observed in callus from mature zygotic embryos, low ABA concentrations increased number of somatic embryos.[48]
Nodal segments1/2 MS supplemented with different concentrations of BAP, NAA, and GA30.050 mg L−1 BAP achieved optimal bud proliferation + 0.50 mg L−1 NAA, while ½ MS media supplemented with 0.2−1 and 0.10 mg L−1 GA3 + 0.10 mg L−1 BAP showed highest shoot elongation.[49]
Nodal segmentsMS media without PGRsMedia free from GA3 + BAP resulted in best shoot elongation, and WPM + 0.05 mg L−1 NAA + 0.5 mg L−1 BAP resulted in maximum axillary bud proliferation.[50]
Nodal segments½ MS media, De-Fossard Medium, 0.9 µg L−1 BA, 0.5 µM NAAThe best multiplication rate (2.25) was achieved, and 93% of the plants survived acclimatization.[51]
Nodal segmentsMS media supplemented with 0.05 μM NAA, 0.4 μM BA, 1 mg L−1 nicotinic acid, 1 mg L−1 pyridoxine-HCl, 1 mg L−1 thiamine, 2 mg L−1 glycine, 50 mg L−1 myo-inositol, and 30 g L−1 sucroseEndogenous rhythms cause time-related fluctuations, resulting in rooting variations among closely related genotypes.[52]
Epicotyl segments½ MS supplemented with 1/6× CaCl2, 2% (w/v) sucroseAuxin reduced mean rooting time, and light conditions did not affect the rooting efficiency; with increased age, decreased rooting capability was observed.[53]
Axillary buds½ MS supplemented with 1 g L−1 ABAEncapsulation by calcium alginate and storing under low light intensities resulted in the preservation of cultures for up to 3 months without affecting their viability.[54]
Apical shootsMS media supplemented with 0.04 mg L−1 BA, 1% sucrose, with/without charcoal38–85% survival was observed with plants exposed to PSV2 for 30 min in liquid nitrogen.[55]
Nodal segmentsMS media supplemented with 30 g L−1 sucroseThe best in vitro establishment, multiplication, shooting, and rooting were achieved using red–blue LEDs and fluorescent lamps.[56]
Abbreviations: 2,4-D—2,4-dichlorophenoxyacetic acid; ABA—abscisic acid; BA—6-benzyladenine; BAP—6-benzylamino purine; CaCl2—calcium chloride; CO2—carbon dioxide; Cu—copper; DTE/DTT—dithioerythritol; DKW—Driver and Kuniyaki Walnut; EDM—E. dunnii basal medium; Fe—iron; GA3—gibberellic acid; HCl—hydrochloric acid; IAA—indole-3-acetic acid; IBA-indole-3-butyric acid; JADS—Juan, Antonio, Diva, and Silvian; K—potassium; LED—light-emitting diode; Mn—manganese; NAA—α-naphthaleneacetic acid; PGRs—plant growth regulators; PSV2—plant vitrification solution 2; PVP—polyvinylpyrrolidone; PVPP—polyvinylpolypyrrolidone; QL—Quoirin and Lepoivre; S—sulfur; SH—Schenk and Hildebrandt; TDZ—thidiazuron; WPM—Woody Plant Medium; Zn—zinc.
Table 2. The table below highlights the composition of the media, additives, and sterilants used in various successful attempts regarding Eucalyptus hybrid production.
Table 2. The table below highlights the composition of the media, additives, and sterilants used in various successful attempts regarding Eucalyptus hybrid production.
SpeciesExplantSterilantMedia; PGR (If Any)Area Studied and Scope of WorkReferences
E. camaldulensis × E. tereticornis and E. torelliana × E. citriodoraNodal segments from mature trees (30–32 yrs)0.15% HgCl2MS media, WPM, SH medium, B5 medium; BAP, NAAHybridization of Eucalyptus species. The study reported that two hybrids developed that showed superior performance than parental genotypes.[17]
E. grandis × E. nitensAxial buds10 g L−1 CaOClMS media; BAP, IAA, metatopolin, kinetinIndividual evaluation of each stage of micropropagation. The study reported that Auxins are principal components of media, and expression of different auxin transporters might be used as markers to identify Eucalyptus spp. amenable for micropropagation.[29]
E. erythronema × E. stricklandiiSeedlings germinated in vitro3% NaOClMS media supplemented with sucrose 30 g L−1; IBA, NAA, Gibberellic acidsFirst micropropagation report of ornamental Eucalyptus spp. The study reported that successful micropropagation from juvenile seedlings was achieved.[44]
E. benthamii × E. dunniNodal segments from 1-year-old plantsNaOCl½ strength MS media; PVP40, NAA, BAPOptimization of chlorine concentration for explant sterilization and optimum ratio of PGRs for shoot elongation. The study reported that 0.5% NaOCl is suggested for nodal segments; 0.50 mg L−1 BAP + 0.05 mg L−1 NAA provides the highest number of bud proliferation.[49]
E. erythronema × E. stricklandiiNodal segments1% NaOClMS media supplemented with sucrose 30 g L−1; 0.05 μM NAA and 2.22 μM BAPEffect of different light intensities on micropropagation efficiency. The study reported that red–blue LEDs and fluorescent light result in higher vigor, high photosynthesis, and increased shoot and root proliferation.[56]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Sharma, V.; Ankita; Karnwal, A.; Sharma, S.; Kamal, B.; Jadon, V.S.; Gupta, S.; Sivanasen, I. A Comprehensive Review Uncovering the Challenges and Advancements in the In Vitro Propagation of Eucalyptus Plantations. Plants 2023, 12, 3018.

AMA Style

Sharma V, Ankita, Karnwal A, Sharma S, Kamal B, Jadon VS, Gupta S, Sivanasen I. A Comprehensive Review Uncovering the Challenges and Advancements in the In Vitro Propagation of Eucalyptus Plantations. Plants. 2023; 12(17):3018.

Chicago/Turabian Style

Sharma, Vikas, Ankita, Arun Karnwal, Shivika Sharma, Barkha Kamal, Vikash S. Jadon, Sanjay Gupta, and Iyyakkannu Sivanasen. 2023. "A Comprehensive Review Uncovering the Challenges and Advancements in the In Vitro Propagation of Eucalyptus Plantations" Plants 12, no. 17: 3018.

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop