Critical Review on Plant Micropropagation of Ethiopian Plants Reported So Far: Existing Gaps, Required Standardization, and Future Research Direction

Plant micropropagation research in Ethiopia requires concerted efforts to meet desired levels of application for sustainable utilization of the country’s diverse plant genetic resources. The purpose of the present review is to provide an update on the results of plant micropropagation conducted so far in Ethiopia. It assessed their strengths and identified gaps in order to standardize research methods and indicate future research directions. Two cereals, three oil crops, three spices, five medicinal plants, two high-value crops, six fruit plants, nine root crops, and one endangered multipurpose shrub were reviewed. The assessment of previously published research was carried out in terms of methods used in the selection of ex plants and their disinfestations, culture vessels, and media used with a variety of combinations and concentrations of plant growth regulators, macro- and micronutrient requirements, culture environments, and genetic stability of regenerated plantlets. Further assessments include the utilization of plant growth-promoting microbes and applications of “omics” research in order to establish standardized, efficient, and cost-effective micropropagation techniques. The findings of the assessments are summarized and current advances are highlighted, along with recommendations for future plant micropropagation studies in the country.


Introduction
Plant micropropagation is one of the applied biotechnology tools that have been utilized in horticulture, forestry, plant breeding, conservation, and disease-free plant production. Its utility can be expanded to culture-based in vitro cloning, the production of bioactive compounds, secondary metabolites, and engineered molecules such as vaccines and multiple pharmaceuticals [1].
Micropropagation is essential in the understanding of somatic embryogenesis [2], haploid plant production [3], and provision of experimental systems for physiological, proteomics, transcriptomics, and epigenetic analysis to study plant cell "dedi erentiation" [4][5][6][7] or transdi erentiation. Standardization of plant micropropagation impacts current advances in biochemistry, genetics, and cell biology through which applications of micropropagation can be widened to improve quality, yield, disease resistance, and stress tolerance by editing and selecting genes and genomic regions [1,2]. ese expensive applications and their detailed processes require standardization, repeatability, and speci cations for sustainable utilization of plant resources. Such detailed processes of standardization can be exempli ed by various steps followed to solve problems of optimization in meristem culture for disease-free plant production and cryopreservation for the conservation of recalcitrant species like co ee.
Plant micropropagation for various applications has various challenges and is usually addressed through di erent approaches, including through appropriate selection of ex plants and their proper disinfection, culture media optimization, as well as the utilization of different types of nitrogen and carbon sources, growth regulators, macronutrients, micronutrients, and culture environments. Other modern approaches that can help alleviate the challenges of plant micropropagation include methods to test for genetic stability, application of plant growth-promoting microbes (PGPMs), and current advances in genomics, transcriptomics, and proteomics. e critical evaluation of the methods used and the results derived from each of these methods would provide crucial information that would lead to the design of cost-effective and repeatable research directions and protocols for the effective utilization of plant genetic resources in the public and private sectors.
In Ethiopia, both the private and public sectors have had a limited number of plant micropropagation protocols for the delivery of disease-free plants [8]. Case studies have reported fifty in vitro protocols across all plant micropropagation laboratories in the country [9]. ese protocols, however, should be further optimized to increase their efficiency and reduction of costs incurred (particularly in hardening in vitro derived plantlets). For the realization of this, it is crucial to have labs equipped with competent staff who can effectively analyze the physiological genetic processes of the micropropagated genotypes that transition from in vitro regeneration to in vivo conditions. Other limitations of plant micropropagation in the country include technical, administrative, financial, laboratory designs, facilities [8,9], and consumables. ese limitations have been exacerbated by poor maintenance of the facilities installed before the required manpower to run and maintain the facilities and also by recent unrest and war in the north and northwest parts of the country.
Many of the current scientific reports on plant micropropagation protocols, including those that are presently peer-reviewed, are not cost-effective, as they were presented in generalized forms without specifying the genotypes used. Avoiding using such inadequately developed protocols or replacing them with appropriate protocols developed for properly defined genotypes adds value to the economical utilization of plant bioresources. Limitations that arise from hasty generalizations due to poorly designed studies and usage of some of the conceptually unclarified terms should also be avoided. Terminologies such as in vitro, in vivo, and plantlet [2,4,6] have been widely accepted and used in plant micropropagation reports. However, terminologies such as totipotent are being challenged currently [6].
Quite often we find issues associated with repeatability, reproducibility, and replicability that impact almost all areas of science [56][57][58]. So these issues are not restricted to plant micropropagation and the societal costs associated with these problems are high [59]. is calls for working out the details of basic design and background factors behind these issues. Some of the societal costs of plant micropropagation culture in Ethiopia can be minimized through the application of plant growth-promoting microbes (PGPMs). However, PGPMs are not currently in use in in vitro plant micropropagation studies on Ethiopian plants. Few studies elsewhere have reported that inoculation with PGPMs in micropropagation [60][61][62] reduces the cost of production of in vitro plants during the micropropagation process due to better survivability and increased resistance to water stress. e present review provides updated literature with an assessment of strengths and gaps in Ethiopian plant micropropagation studies conducted in Ethiopia, in order to show the need for standardized methods for optimal and cost-effective utilization of plant diversity. ese together with future research on the genetic and physiological bases of micropropagation can serve as reliable research directions for the sustainable utilization of existing biodiversity.

Materials and Methods
A survey of published articles and other literature on Ethiopian plant micropropagation studies was carried out to collect data on plant species studied so far. e keywords used in the survey were (1) plant tissue culture research in Ethiopia, (2) plant micropropagation in Ethiopia, and (3) in vitro plant regeneration in Ethiopia.
e Google Scholar branch of the Google search engine was used for searching literature with these key words. e obtained publications were filtered and the most relevant and informative ones were used to generate the data sets presented in Tables 1-3. e surveyed data include objectives of the various studies, reported results, explants used, surface sterilization procedure followed, nutritional and nonnutritional media components used, utilization of different sources of nitrogen, carbon, micronutrients, plant growth regulators (PGRs), type of culture vessel used, in vitro growth and regeneration conditions followed, methods of transfer of propagules from culture to the soil, and genetic stability tests applied (Tables 1-3). e status of the applications of plant growth-promoting microbes for micropropagation was also recorded. e data gathered are organized into distinct but interconnected themes for gap identification and assessments in the discussion session. e discussion is followed by conclusions, current advances, recommendations, and brief highlights about future research directions for plant micropropagation in Ethiopia.

Results Recorded following the Materials and Methods
Described above. e overall stated objectives and results of some of the representative micropropagation studies made so far are provided in Table 3. e stated objectives and results of the various micropropagation studies have wide coverage and include virus cleaning and disease-free plantlet production, screening for salt-tolerant plants, in vitro micropropagation of medicinal and high-value crops, identification of better microtuber induction under various levels of sucrose, optimization of various concentrations of sterilants and plant growth regulators, and in vitro conservations [10][11][12][13][14][15][16][17][18][19][20], [21][22][23][24][25][26][27][28][29][30][31], [32][33][34][35][36][37][38][39][40][41][42], [43][44][45][46][47][48][49][50][51][52][53][54][55]. Other objectives include hormonal and media treatments to overcome hyperhydricity (vitrification) of micropropagated shoots [17]. e studies on plant micropropagation conducted so far in Ethiopia used explants that covered immature embryos, cotyledons, hypocotyls, seeds, lateral buds, nodes, shoot tips, shoot buds, and leaves. e types of surface sterilization agents applied are predominantly 70% v/v ethanol and 1 to 20% w/v sodium hypochlorite. e exposure time to ethanol  Advances in Agriculture was 1 m while it ranged from 5 to 25 m in the case of sodium hypochlorite depending on the species utilized. e exposure time used in the case of sodium hypochlorite did not correlate with the concentration applied. e hypochlorite used was partly an unstandardized local product called "Berekina." e vessels used for culturing were predominately Petri dishes followed by magenta and test tubes. Plant growth regulators (PGRs) and their concentration and tissue culture growing and regeneration conditions differed slightly between the species studied and the explants used. In most cases, the reported studies were conducted with no comparative experimental assessments. In nearly all cases, Murashige and Skoog media was used, but without comparison with other media. e growing conditions and nonnutritional components applied were also similar. Molecular markers were not used for the identification of genotypes studied, and hence, the results reported so far may not be repeated on other genotypes of the same species. Local cultivar names were used as a marker, underestimating the genetic variation that exists within each local cultivar. In most cases, what we call protocol development lacks a comparative assessment. e growth temperature used was predominantly within the range of 25-27°C, and the relative humidity was mostly 70%. However, this information was not provided in some studies. e duration of light hours was predominantly 16 hs, while the light intensity used varied among the various studies. In some cases, the parental ex plants from ecologically different sources were treated as if they were the same [25], thus ignoring the impact of preceding environmental growing conditions on the micropropagation of the genotypes under study. e types of in vitro tissue culture approaches for regeneration and mass propagation of the plant species studied are summarized in Table 3 as callus culture, meristem culture, somatic embryogenesis, and organogenesis. e culture types reported in the studies are mostly for micropropagation except in two cases of somatic embryogenesis [11,24], a single case for unfertilized ovary [43], anther culture [46], callus induction for shoot regeneration [15], and two cases for in vitro germination of seeds and shoot induction [13,23,55].
ese studies identified MS media with some growth regulators individually and/or in combinations for the shoot and root developments [27,[37][38][39][40]42] and determined the minimum number of days for root induction [28], the highest percentage of callus production [29], effect of PGRs on the number of roots [45], and shoot length and shoot fresh weight [27,37,53,55]. e studies also determined the survival rate of micropropagated plants in greenhouses [38,45] and identified responsive explants and polyploidization, for example, to produce larger bulb size in garlic [26], by anticipating an increase in  CI from hypocotyl and cotyledon for in vitro shooting and rooting [40] 4. Carica papaya L. Micropropagation e highest mean, mean number of shoots, leaf, and shoot length were obtained on 1 mg/l BAP and 0.5 mg/l NAA. A minimum (9.5 days) and longer (13.5 days) for shoot initiation were recorded on BAP at 0.5 mg/l + 0.5 mg/l NAA and BAP at 2.0 mg/ l + 0.5 mg/l NAA, respectively. Half-strength MS with 2 mg/L IBA is the most suitable treatment for root induction. e maximum number of roots (16.25) and root length (3.92 cm) were measured on shoots pretreated on MS media supplemented with 1.5 mg/l IBA.
Micropropagation&Optimization of sterilization protocol 5% NaOCl for 10 m is the most effective in surface sterilization. e sterilized seed cultured on ½ B5 media was most suitable with a germination of 26.67%. e highest shoot initiation (89%) of explants produced shoots), the number of shoots per explant, and the number of leaves per shoot were obtained from cotyledonary node explants cultured on MS media supplemented with 2 mg/l BAP within nine weeks. But, the highest shoot length and shoot fresh weight were recorded from control (free BAP) and 6.00 after 42.57 ± 0.58 days of culture BAP, respectively. e highest shoot multiplication (4.56 number of shoot induced) and elongation (2.97 cm) were obtained from the induced shoot cut placed on MS media supplemented with 2.00 mg/l BAP + 6.00 mg/l GA3 and free BAP + 6.00 mg/l of GA3, respectively cotyledon node explant [55] 8 Advances in Agriculture Micropropagation using shoot tips [13] Advances in Agriculture 9 14. Capsicum annuum L.
Micropropagation, conservation Optimatizion of sterilizing protocol 3% active chlorine for 20 m was found to be a better treatment combination yielding 82.5% ± 5.00% contaminantfree germinated seedlings. For shoot induction, MS + 4.5 mg/l B AP + 0.5 mg/l IAA and MS medium containing 8 mg/l Zeatin were found to be better, resulting in 77.5% ± 5.00% and 67.50% ± 5% induction percentage for nodal and shoot tip explants, respectively. Maximum shoot multiplication responses were obtained on MS + 3 mg/l BAP + 2 mg/l Kinetin with a mean number of 9.2 ± 0.2 and 8.6 ± 0.00 shoots for nodal and shoot tip explants respectively.
Best shoot elongation and rooting responses were reported on MS + 0. 5  10 Advances in Agriculture Advances in Agriculture 11 bulb weight and overall productivity. As a continuation of these, the recent studies (2021/2022) covered the effect of MS medium strength, sucrose concentration, and pH level on shoot multiplication and root formation [21], in vitro propagation protocol for rapid regeneration of high-value crops like sugarcane using a completely randomized design with factorial arrangement [30]. In these studies, treatments with growth regulators had an overall positive impact on all parameters measured but with better performance due to growth regulator types, their concentrations, and combinations for the particular plant species studied [34][35][36][37][38][39]. BAP, IBA, IAA, and NAA were the predominantly used plant growth hormones and are followed by 2-4 D, TDZ, IMA, and GA3.  Tables 1-3 was assessed and discussed using the following interconnected thematic factors, and these are followed with conclusions and recommendations for standardized research in plant micropropagation in Ethiopia.

Embryo Morphology, Somatic Embryogenesis, and Networked Gene Regulation on Micropropagation.
e production of functional plants in micropropagation can be increased by improving the morphology of somatic embryos during induction and development through culture manipulation. However, this is not always clear from the reports made so far on the Ethiopian plants. e degree of somatic embryogenesis varies with explants of particular genotypes used and their physiological states and these have gene expression patterns [63,64]. e genotypic variation within a given species affects the process of embryogenesis while the culture manipulation improves the morphology of somatic embryos during induction and development where all are influenced by the identity of genotypes in any standardized study.
Auxin and wound-induced callus formation pathways converge to the same gene regulation network and rely on the cooperative action of defined sets of transcription factors [6,65]. Gain or loss of function of many cell cycle or developmental regulators might result in callus formation [5]. e detailed genetic structure, molecular marker, and allelic types of these networked pathways may differ between species, genotypes, type of explants used, and previous environmental growth conditions of parental stocks. ese impact the standardization and repeatability of micropropagation. In addition to the detailed genetic structures, phenomics and allelic oscillation can limit the results of any micropropagation studies. us, integrating "omics" data at broader levels of systems biology through using genomics and phenomics could result in a better prediction of micropropagation. us, phenomics and other omics studies should be pursued on genotypes of interest to enable a standardized protocol development of plant micropropagation.
Reports in the literature show that rooting capacity differs due to endogenous auxins and ranges from very easy to very difficult to root [66][67][68]. ese differential capacities could be due to differences in gene regulation and their networks. us, plant micropropagation and regeneration studies in Ethiopia need to take into account this for each genotype using appropriate markers and, when possible, with information on variant genes that respond to endogenous auxins. e seasonal variation and variation in nutrient use efficiency and the age of the stocks or mother explants also impact micropropagation and should be defined and stated unlike the various reports summarized in Tables 1-3.

Reliable Genetic Stability Tests on Micropropagated
Propagules. e micropropagation protocols described in Tables 1 and 2 were expected to have a high frequency of organogenesis or embryogenesis, with propagated clones remaining true to their parent types when tested using reliable genetic markers. e methods employed to assess the genetic stability of plant genotypes in the past included morphology [69], secondary compounds, protein electrophoresis [70,71], and cytology [72,73]. Other methods focused on chromosomal variation, restriction fragment polymorphism (RFLP), intersimple sequence repeat (ISSR), simple sequence repeat (SSR), and single nucleotide polymorphism (SNP) markers-based analyses. Any of these methods that assess genetic stability were not applied in the studies whose results were summarized in Tables 1-3. e micropropagation reports assumed a low risk of genetic instability and failed to consider the minimum standardized DNA markers [74] like the one developed and reported for Magnolia sirindhorniae Noot. & Chalermglin, which confirmed the genetic uniformity and stability of regenerated plants of M. sirindhorniae.
When proteins are used as markers, attention should be given to the stages of growth and physiology of the plants used. Factors that control the expression of proteins through differential levels of transcription in various organs do differ even in genetically identical plants and thus need to be taken cautiously. Comparisons based on these characters are rather difficult, particularly when data from different geographical areas [70,71] and physiological stages are considered. Significant alterations, in both chromosome number and morphology, have been demonstrated as a result of regenerating plants from culture via a callus phase [73]. Observations of these changes in micropropagated plantlets have not yet been tested in the plants reported in Tables 1-3. However, one should note that the incidence of gross chromosomal aberrations and their progeny is overemphasized since chromosomal abnormalities are usually eliminated in the plantlets [73]. When required, such tests should be done on different sections of plants since some plants may be chimerical and possess sectors with different ploidy levels higher in cell or callus cultures than in plants regenerated directly from preexisting meristems.

Culture Environment for Micropropagation and Establishment of Propagules in the Field.
e micropropagated plants should be protected from epigenetic variation that results from environmental factors and various stresses including doses of PGR and levels of humidity in culture vessels [75][76][77]. In most of the reported cases of the present review, optimized establishment of propagules in the field is not included. ey did not provide clear and complete information on sources, history, and genotypes of explants used. e components of culture medium and their interaction as well as the timing of the subculture period are not provided. e reported in vitro micropropagation growth conditions comprised temperature, light, pH, and growth culture vessels, each of which varies among the plants studied.
(i) Temperature: incubation temperature for micropropagation ranges from 20 to 28°C [78]. Some species root more effectively at higher temperatures.

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For example, some cultures of grapes root better when exposed to higher temperatures. A study on grape micropropagation for salt tolerance in Ethiopia (Table 1) used a temperature of 27°C. However, it is not clear whether the difference in temperature between reports from other studies and that of the reports from Ethiopia [38] is due to differences in genotypes used and/or other factors. Fluctuations in temperature during day and night influence growth and organogenesis in cultures [79], and this suggests the need to redefine the optimum temperature requirements of the various reports in Tables 1 and 2, together with various sources of light given below. (ii) Light: the role of light (irradiance, spectral quality, and photoperiod) is probably one of the most neglected and poorly studied factors in plant tissue culture including micropropagation studies and is not limited to those reported in Tables 1 and 2. Light quality should be considered when choosing light sources for culture rooms, and both quality and intensity should be checked periodically. In addition, a laboratory with an appropriate design and regular electric supply is required. In embryo-derived callus cultures of Douglas fir, red light stimulated adventitious bud formation [80,81], although this is not universal. For example, in tobacco callus cultures, blue light is more effective than red in stimulating shoot growth [82]. e effect of light on callus induction/morphogenesis/somatic embryogenesis differs between species and genotypes used and thus needs to be defined through experiments. In some reported cases, dark pretreatment was considered helpful. is was shown in an experiment on Cicer arietinum using explants excised from leaflets of immature plants grown under 24 hours of darkness with better callus initiation response than the same explants grown under a 16-hour photoperiod [82]. (iii) pH: generally, cell growth in culture is not significantly affected under a broad range of initial pH (4.0-7.0) of the medium. It is difficult to draw meaningful conclusions about the optimum pH of a medium unless pH is constantly monitored and readjusted. e pH of the medium is influenced by factors, such as the genotype of an explant, media type, initial pH, and incubation time. Woldeyes et al. [21] reported that both shoot multiplication and root formation of okra were reduced with decreasing pH and resulted in no root formation at pH 5.0. However, the fluctuation of the pH during the experiment was not monitored, and the genotype used was unmarked. e decrease or increase in pH reported elsewhere depends on whether the media used contain ammonium or nitrate as a nitrogen source [83,84]. Hence, information on a range of pH needs to be defined with nitrogen sources in the media used on genetically marked genotypes in order to optimize and develop a repeatable protocol of micropropagation. (iv) Growth media culture vessels used: for media cultures, Petri dishes, Erlenmeyer flasks, bottles, magenta vessels, or test tubes are commonly used, and each of them affects growth differently. e volume of both the atmosphere [79] and nutrients in a vessel affects growth rates and morphogenesis and thus should be standardized. Proper ventilation of the atmosphere in the vessels is important since the accumulation of volatiles or ethylene production causes vitrification and inhibits morphogenesis [85] and other physiological processes. Rapid changes in the atmosphere, for example, when vessels are opened, may result in a temporary "gas shock" of the culture [86]. Studies similar to those summarized in Tables 1-3 as well as other studies with similar objectives should evaluate the quality of micropropagation and regeneration in test tubes, magenta vessels, and Petri dishes (with their specified closures that permit air exchange) with constant and variable volumes of media.

e Problems of Defining Precultures and Physiological
Stages of Explants. Immature tissues and organs are invariably more morphogenetically plastic in vitro than mature tissues and organs and thus have a high frequency of cultural survival and growth rates in vitro. However, the various reports given in Tables 1-3 failed to define and standardize species and genotype-dependent physiological stages for optimum micropropagation. In some studies like the case of noug (Guizotia abyssinica), the results obtained were not compared with other similar studies [87]. e differences may be due to the types of genotypes and physiological stages of explants used. Cultural conditions of explants and background information on environmental growth conditions of parents and other prior treatments of the species to study should not be overlooked unlike studies reported on Ximenia americana L. [25] in Ethiopia. ese authors [25] used X. americana samples from two ecological sources (Boset in the central-eastern Oromia and Gambela regions in Ethiopia) but reported their results without treating the two sources separately.

Surface Sterilization of Explants.
e most common surface sterilants in plant micropropagation culture are calcium and sodium hypochlorite, with calcium salt being less toxic to tissues than sodium salt [88]. However, calcium hypochlorite reacts with carbon dioxide in the atmosphere and is chemically unstable. Most commonly, a dilute solution of sodium hypochlorite (0.25-2.63%) is used as a disinfectant. An emulsifier such as Tween-20 (polyoxyethylene sorbitan monolaurate) is added at the rate of 1 drop per 100 ml of solution.
e most commonly used sterilization agents on Ethiopian plants reported in Tables 1 and 2 were ethanol and sodium hypochlorite. e concentrations of sodium hypochlorite used range from 1 to 20% w/v with exposure times of 5-20 minutes. However, the exposure time and concentration were not correlated. Besides, all hypochlorite should have been completely rinsed out from the tissues, because any trace left behind will interfere with amino acid uptake and metabolism [88]. However, none of the reports summarized in Tables 1 and 2 carried out tests for traces of hypochlorite left after washing with water. ere were no comparative assessments of different surface sterilants except in a few cases, like in a study [34] that examined the in vitro propagation protocol using a completely randomized design with four replications and a factorial arrangement for the rapid regeneration of apple rootstock and scion node culture. e results of sterilization using 0.3% mercuric chloride for 7 min scored a higher percentage of no contamination although this also varied among genotypes studied. As a special case of laboratory conditions, bacteria and fungi, may escape surface sterilization of original explants and arise as laboratory contaminants [89]. ese factors should have been taken into consideration along with the type of explants, disinfectant type, and/or treatment time employed.

Nutritional and Nonnutritional Composition of Medium.
e majority of plant micropropagation media are chemically defined with additional variables of plant growth regulators, the concentration of agar, and methods of preparation [90,91]. White media [92] contains the nutrients normally required by plant cells, especially for root cultures. However, the amounts of nitrogen and potassium were found to be inadequate to sustain maximum growth of callus and cell suspension cultures [93,94]. Callus and cell cultures of some species prefer MS, while others grow better in the B5 Gamborg et al. [95] or Erikson [96] media. e need for richer mineral salt mixtures is compensated by adding yeast extracts, protein hydrolysates, amino acids, coconut milk, or other organic supplements. ere is a wide use of the MS [97] medium or its modifications. e MS and Erikson [98] media are similar, but the Erikson media contain twice the amount of phosphate and much lower concentrations of micronutrients than the MS medium. e B5 Gamborg et al. [98] medium contains relatively low amounts of ammonium, a nutrient that may repress growth in batch cultures. A number of culture media have been developed and tested to satisfy the needs of a variety of plant cells and tissues for micropropagation. Some of these media differ in degree as dilutions of standard media, while others combine the micronutrient elements of one with the micronutrient elements of another [99]. e Schenk and Hildebrandt (SH) [100] medium resembles B5 Gamborg et al. [98]. However, the amount of mineral salts is slightly higher, and ammonium and phosphate are supplied as NH 4 H 2 PO 4 . e salt content of the Heller medium (Heller, 1953) cited in [101] is relatively low. Different concentrations of vitamins, amino acids, Myoinositol, and phytohormones are usually added to the chemical composition of the various media to meet the optimum growth requirements.
ere are also numerous other media in use, including Litvay, Anderson, Quoirin-Lepoivre, Durzan, Woody Plant Medium (WPM), Gresshoff-Doy, and Zimmerman media. A major difference between MS and WPM is the level of macronutrients. ere is a wealth of literature on various types of media and additives; however, in Tables 1 and 2, the media composition used is mainly MS with no comparison with other media for a likely better performance. Agar is among nonnutritional media components and sometimes inhibits shoot growth. Substituting agar with liquid or gelrite-solidified media strongly promotes the growth of shoot cultures. A liquid or gelrite-solidified media increases succulence and vitreous growth [102], which is vulnerable to mechanical damage. In some cases, a compromise can be achieved by using a combination of agar and gelrite or using charcoal to reduce the inhibitory effect of agar with the removal of any cellular waste products. is aspect of the work is seldom covered on Ethiopian plants studied so far. Morphogenesis as well as callus growth rates and growth response of the cultures partly depends on the concentration and degree of purification of agar [90,103]. Agar is a source of many minerals, in particular, sodium and possibly some vitamins and toxins, which may complicate the metabolic and nutritional requirements of micropropagation culture studies. e most interesting agar substitutes studied include positively charged dextran microspheres, plant agar, a starch copolymer, poly acryl amide, silica, and Ficoll, a sucrose polymer. Activated charcoal adsorbs and prevents the browning of tissues [104] and stimulates embryogenesis and rooting. However, the type and purification of agar and activated charcoal added to nutrient media to remove aromatic waste products excreted by cultured tissues are not given in most of the reported cases summarized in Tables 1 and 2.

Sources of Nitrogen, Carbohydrates, Micronutrients, and Plant Growth Regulators in Plant Micropropagation
Culture Media 3.7.1. Nitrogen. Most of the micropropagation studies conducted on Ethiopian plants so far lack comparative studies to identify referred nitrogen sources. e growth rate in the medium containing nitrate is lower than the rate in the medium containing both nitrate and ammonium. On the other hand, the growth on ammonium in excess of 8 mM can be deleterious and is generally considered toxic to plant cells [98]. Most plant micropropagation media incorporate both nitrate and ammonium salts as an inorganic nitrogen source for growth. Embryogenesis of some species like carrot is severely inhibited when grown on media containing nitrate as the sole nitrogen source, while the addition of low concentrations of ammonium stimulates both growth and embryogenesis [105]. An adequate assessment of the suitability of nitrogen sources in a medium and working out the genes involved in nitrogen metabolism and embryogenesis are needed for the required standardization and repeatability.
Advances in Agriculture 3.7.2. Carbohydrates. In addition to serving as carbon and energy sources, carbohydrates play an osmoregulatory role (as osmoticum) and are in situ regulators of morphogenesis in both a medium and tissue. us, many micropropagation cultures, especially those that involve embryo cultures, perform properly only when cultured on a nutrient medium with a high osmotic potential [106]. Sucrose is generally the best carbon and energy source [106] and has been the carbohydrate of choice in the vast majority of reports on shoot induction and micropropagation. In many plant species, sucrose is used at a final concentration of 10 g/L to 30 g/L. However, it is not always the most effective carbohydrate. Fructose, glucose, sorbitol, and mannitol were found to be effective in the shoot initiation and micropropagation of several plants in a stage-specific fashion. Myo-inositol may not be essential but is added since it has been shown to enhance callus growth. Other studies have shown that the generation and establishment of photoautotrophic cultures require cell selection and manipulation of levels of growth regulators and carbon dioxide [107]. Several studies have indicated that the endogenous carbohydrate status of the explant at the time of selection is important in the development of adventitious roots. e role of various carbohydrates, their optimum concentration, and endogenous status in explants need to be studied to fill the gap left in studies on Ethiopian plants reported in Tables 1 and 2, as well as those that will be studied in the future.

Micronutrients.
e optimal requirements of micronutrients, including their components and how they are dissolved, for the species summarized in Tables 1 and 2 need to be revisited. Iron chelates stimulated embryogenesis and root growth, whereas iron dissolved in nonchelated form did not. EDTA, without iron, at concentrations comparable to those used in micropropagation culture media, stimulates nitrate reductase [108] and inhibits ethylene formation and this may be significant because reduced nitrogen stimulates embryogenesis, while ethylene inhibits it thus negatively impacting micropropagation. e optimum level of micronutrients and their combined effect are thus of interest to work out in the plants reported.

Plant Growth Regulators.
e effects of auxin type, concentration, and their interactions with each other and with other PGRs influence in vitro plant regeneration and micropropagation. Bonneau et al. [109] tested the effect of 200 growth regulator combinations in culture media on the European Spindle Tree (Euonymus europaeus L.). e results indicated that only four combinations (IAA-benzyl amino purine and IAA-kinetin) allowed differentiation of somatic embryos depending on auxin exposure time. However, this differs among various species, genotypes, and explants used. Amente and Feyissa [30] used a completely randomized design and factorial treatment arrangements (unlike many other reports) of different levels of BAP for in vitro propagation of sugarcane in Ethiopia to enhance the availability of healthy and true-to-type planting materials. However, the claim that the parent plants were true to type was not tested and reported and needs further study.
All levels of hormone treatments used in Ethiopian plant micropropagation culture studies had positive impacts on all parameters measured [30][31][32][33][34][35][36][37][38][39]. However, only specific hormone types and concentrations resulted in better performance during plantlet acclimatization. Genotype differences in the species studied evoke varying responses to the same PGRs where hormone type and concentration are considered for effective in vitro propagation of respected genotypes/cultivars. e presence of cytokinins, kinetin, and/or zeatin in the induction medium for the expression of somatic embryogenesis and micropropagation needs to be worked out with appropriate design and factorial treatment arrangements for each specific genotype of a given plant species. In addition to this, the role of ABA to stimulate callus formation and embryogenesis and the application of silver nitrate to enhance embryogenesis for micropropagation in species with high ethylene levels need to be studied and documented.
Some plants with relatively high concentrations of auxins induce the regeneration of roots due to interconnected distribution and signaling profiles that stimulate cambial activity and align with root apical meristems [63,64]. Rooting of adventitious shoots and cotyledon cultures, and the regenerated shoots from callus in some angiosperms, can be promoted by low concentrations of auxins [64,110]. Hence, the removal of an auxin from the medium, leads to root formation. e role of different auxins and their levels of performance for micropropagation of the various species reported in the present review remains to be thoroughly worked out. e distribution of auxins in various meristems is well established as presented above. e physiological effects of each PGR are determined by the kind of growth regulator and its concentration, the presence or absence of other PGRs, and the genetic makeup and physiological status of the target tissue in a micropropagation. We do not yet have data on these factors in many species, including in those reported in Tables 1 and 2.
Other studies that are of interest could be the effect of growth hormones, colchicine concentration, and immersion time [26] for increasing ploidy levels followed by micropropagation in several economically important bulb bearing plants.

Application of Plant Growth-Promoting Microbes (PGPMs) in Micropropagation.
Propagules developed under aseptic conditions are transferred to soil by protecting them from soil pathogens through the application of special antifungal treatments. In addition, growing them in special transplanting media increases their survival, which is influenced by the kind of substrates used. For example, the percentage of survival of Cicer arietinum propagules was high (85.4%) in pure vermiculite than in a mixture of vermiculite and perlite (1 : 1) (20.4%). e propagules transplanted into sandy soil and sawdust mixture (1 : 1) did not survive [82]. A consolidated effort is required in optimizing diverse forms of micropropagation from various 16 Advances in Agriculture sources of explants and such efforts should include specifications of appropriate transplanting media in various ecologies. PGPMs help plants to access nutrients and protect them from diseases, pests, and abiotic stresses. is is done through a variety of processes, including the production of phytohormones, siderophores, phosphate solubilization, and induction of plant intrinsic systemic resistance responses [111]. Mengistie and Awlachew [112] reported indigenous Bacillus species as PGPR for different varieties of tomato under ex vitro (not through in vitro plant micropropagation) and found promising results for the conditions in Northwest Ethiopia. PGPMs as sustainable plant growth enhancers have the potential of addressing multiple stresses and thus call for them to be integrated with research in plant breeding, micropropagation, and agronomic improvements. Soumare et al. [111] presented an overview of the importance of PGPMs and their potential applications in plant micropropagation where their analysis, based on published articles, revealed that the process of in vitro classical tissue culture techniques under strictly aseptic conditions needs to be reviewed. PGPMs can positively impact the growth of explants and ensure better survival by sustaining the shock during transplantation into a greenhouse or glasshouse and field [112]. Plants from micropropagation are adversely affected by water stress, because of the low absorption capacity of their roots. Inoculation of these plants with PGPMs in vitro is an effective step to deal with low water absorption capacity as well as to boost posttransplant performance of in vitro grown plants through increasing nutrient availability and inducing resistance to pathogens.
Mycorrhizal fungi also produce hormones that control plant development and activate signaling pathways during biotic and abiotic stresses. Meixner et al. [113] showed that plants inoculated with AMF (Arbuscular Mycorrhizal Fungi) had higher levels of auxins than noninoculated plants. Fungi, especially AMF, play an important role in water uptake and availability [114], thereby increasing the rate of photosynthesis and osmotic adjustment under environmental stresses [115]. AMF also increases the uptake of micronutrients such as P, Zn, Cu, and Fe. e contribution of both AMF and PGPMs is significant during the acclimatization phase because the weak adventitious root system (without root hair) of in vitro plants does not allow optimal absorption of nutrients from the soil during the early stage of the weaning step. e lower survival rate and poor establishment of in vitro plants under field conditions may also be due to the fact that the transferred in vitro plants do not find their natural microsymbiont partner. Diez et al. [116] showed that in vitro mycorrhization with Pisolithus tinctorius and Scleroderma polyrhizum strains increased the formation of secondary roots. Similarly, Sahay and Varma [117] reported a 90% posttransplantation survival rate of micropropagated tobacco and brinjal plants treated with the endophytic fungus, Piriformospora indica. is biopriming has also been reported to increase resistance against pathogen attacks [118]. Nevertheless, certain endophytic fungi can be plant pathogens like the case with Fusarium equiseti, which was suspected to cause bamboo blight and culm rot diseases and limit the micropropagation process [119]. e use of the mycorrhization technique can be important for the growth and development of micropropagated plantlets; however, prior to this, the process of production of pure fungal inoculum for micropropagation needs to be resolved.
Kargapolova et al. [120] have shown the efficacy of the inoculation with Ochrobactrum cytisi in potato micropropagation. A 50% increase in the mitotic index of root meristem cells and a 34% increase in shoot length were reported under ex vitro conditions. In vitro mycorrhization of micropropagated plants before acclimatization increases survival and resistance to water stress and ensures better mineral nutrition of the plant by enhancing the functionality of the root system [121]. Numerous findings validated the use of in vitro mycorrhization techniques in several plant species, such as Castanea sativa [122], Helianthemum spp. [122], Citrus spp. [123], and Quercus suber [116]. Research is needed to select efficient, multifunctional, stress-tolerant PGR-producing microbes that have ecological plasticity for use at different stages of micropropagation. Particular attention should be given to mixed-strain consortiums rather than monostrain inoculums to take advantage of functional complementarities to be carried along with plant micropropagation studies in Ethiopia. A great deal of effort should be devoted to the bioformulation of these microbes for suitable applications. e use of nanoformulations may enhance the stability of biofertilizers [124,125] with respect to heat, desiccation, and UV inactivation. e need for bionanotechnology research inputs in in vitro micropropagation culture techniques is obvious. e use of microbes deserves careful monitoring of endophytic communities, especially for plants used as raw food because some strains that are pathogenic to humans can be stably maintained in cultivated tissues and ex vitro plants. In addition, much remains to be learned from PGPMs in order to identify appropriate candidates to develop bioformulations for suitable application in plant micropropagation. Studies on the responses of crops and other economically useful plants to inoculation with symbiotic and nonsymbiotic PGPMs will help identify which plants in the Ethiopian plant bioresources are suitable candidates for microplant biotization. Studies to identify and evaluate the capacity of micropropagation studies using PGPMs should be seriously considered as discussed above. Application of inoculants of plant growth-promoting microorganisms (PGPM) on the plants reported so far through micropropagation reduces costs incurred during the whole micropropagation process and transfer to transplanting media.

Highlights of Advances in Plant Micropropagation Studies.
Plant micropropagation benefits from progress in several research fields, including emerging and cutting-edge biosciences. Such integrated advances in science enhance conservation, mass propagation, genetic manipulation, bioactive compound production, and crop improvement [125,126]. Other areas of plant micropropagation technology that should show advances in Ethiopia include plant Advances in Agriculture genetic resource management and regeneration of endangered plant species through recombinant DNA technology and genetic engineering for the safe conservation of their genetic stock in gene banks (cryopreservation) as suggested by Kumari [126]. Other additional advances should cover epigenetics, transcriptomics, and proteomics-based testing for the plasticity of genome responses as well as for the application of plant growth-promoting microbes in micropropagation research programs.
Advances in plant micropropagation offer economic prosperity by drawing the attention of applied biologists, businessmen, businesswomen, and entrepreneurs. Such prospecting advances should be based on the reorganization of the currently dispersed plant micropropagation and related laboratories into research program laboratories equipped with the necessary capacity and capabilities for conducting research on commercially high-value plants and for the production of phytochemicals for commercial purposes. A laboratory of this type must meet a high standard of cleanliness with regular fumigation, air filtering, standardized electrical wiring safety, power backups, and ventilators, which are seldom found in many existing laboratories in the country.

Major Conclusions, Future Prospects,
and Recommendations e objective of this paper is to review and assess the various factors considered in micropropagation in the so far reported studies on Ethiopian plant species, with reference to the results and efforts made in the country, the challenges faced, and the major gaps noted in order to propose recommendations regarding standard micropropagation. e results and growth conditions reported may be difficult to repeat on other genotypes due to the lack of specificity of the genotypes used and their markers.
In order to maintain clarity, repeatability, and enrichment of current biological advances, recent advances in proteomics, transcriptomics, and epigenetic analyses should be anchored in micropropagation studies. In most cases, the plant micropropagation studies reported to date underrate the importance of explants, the culture environment, timing (subculture period, dosage of PGR), and interactions between these factors. A proper physiological stage of development for optimum regeneration has to be established and standardized in terms of the species used and the specific genotype selected.
Comparative assessments of surface sterilants require studies on methods of distribution of disinfectants and adequate disinfection of explants. In order to determine the most appropriate media and agar type for Ethiopian plants, MS media should be compared with various other media.
ere should be an adequate assessment of both macronutrients and micronutrients and their combined effects, for specific species and genotypes that are properly identified and marked in order to ensure the genetic stability of micropropagated plants.
Air exchange in culture flasks needs to be studied with appropriate design. It is useful to define the information of light quality and intensity and dark pretreatment on the Ethiopian plants/crops so far reported and others to be dealt with in the future. PGPMs should be applied to select efficient, multifunctional, stress-tolerant, and PGRs-producing microbes that have ecological plasticity for their use in different stages of micropropagation, with particular attention to mixed-strain consortiums to take advantage of functional complementarities.
Micropropagation studies in Ethiopia require a standardization of laboratory design and facilities in order to get the maximum benefit from existing useful plants. Studies on plant micropropagation cultures must be programmatic and have the necessary levels of skilled human resources, technology, infrastructure, and institutional structures to contribute effectively to competitive agricultural research programs. In order to improve crop resistance to biotic and abiotic stresses, as well as to increase food crop nutritional quality, this technology should be developed. Furthermore, these programs should take into account the impact of various systems of agroecological diversity in the country.
Research programs in plant micropropagation and related studies should be focused on the current needs of the country without compromising opportunities that may arise in the near future or beyond. A sound research program should be set up to develop products of economic importance that are sustainable and reap the benefits of the country's abundant plant bioresources. In the use of plant micropropagation, one should consider indigenous plants with high economic potential that are underutilized, while also integrating various scales of indigenous knowledge.

Data Availability
is MS is a critical review based on the published reports that are referred and listed in the reference lists of the MS, Data are thus available as a secondary document.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
e first author designed the study, analyzed the data, and wrote the paper. e second author collected the data and the required literature and contributed to the writing. e third author commented, edited, and proofread the prepared manuscript. e corresponding author responded to the queries raised by the reviewers, edited, and contributed to the overall interpretation of the data collected. All authors read and approved the final manuscript.