The process of micropropagation generally comprises four stages irrespective of the technique used: (1) establishment of aseptic culture to remove microorganisms, (2) shoot induction and multiplication to obtain larger number of shoots, (3) root induction to obtain a complete rooted plantlet and (4) acclimatisation of rooted plants to train them to withstand the harsh outside environment ^[1][2][3][19,55,69]. Intact plant regeneration of Duboisia was first achieved in 1980 ^[4][90] and there have been several advances in micropropagation over the last four decades.

The first step in any micropropagation protocol is to treat the explant material using various chemical treatments and a series of washing steps to remove any microbial contamination to establish an aseptic culture. In addition to the chemicals, the type of explant, material collection season/time and health of the mother plant are important factors to consider for successful clean culture establishment ^[5][91]. Generally, the mother plants are maintained in containment under a glasshouse or controlled condition with strict pest disease management strategies in place, such as application of antibiotics and fungicides ^[6][62]. In establishing clean cultures of Duboisia, different sterilisation protocols have been reported previously (Table 1). Comparing the contamination level of different Duboisia explant sources, Lin ^[1][19] showed that shoot tips are comparatively difficult to disinfect compared to young leaves, seeds or seedlings. They recorded 100%, 97%, 52% and 31% clean culture establishment with seedlings, seeds, young leaves and shoot tips, respectively. In the same study, explants collected during the later spring season were found to be more difficult to sterilise (clean culture 26%) compared to winter material (clean culture 40.7%). This was attributed to the microorganism suppression effect due to the low temperature during winter periods compared to warmer and more humid summer months.

Shoot regeneration under in vitro conditions is determined by several factors. This includes the quality and the type of explant, culture media composition and culture incubation conditions ^[12][13][93,94]. Most often, different species of plants have specific requirements with respect to the factors mentioned above, but in the case of most woody perennials, there are cultivar-specific requirements for culture media composition, which have been well documented for peach ^[14][95], apple ^[15][96] and avocado ^[2][55]. Physiological maturity is also an important determinant for shoot regeneration in culture. Juvenile tissues (before flowering stage) are reported to have a higher potential for in vitro regeneration compared to mature material (after flowering) ^[16][97], particularly for woody species ^[17][18][98,99]. With respect to Duboisia species, young seed and seedling explants exhibit a faster response and higher shoot induction frequency than shoot tips and young leaves, which have been shown to produce calli with better quality, benefitting indirect shoot regeneration ^[1][19].

It can be shown that appropriate culture media promote healthy and quality growth of the culture, while unsuitable media adversely inhibit culture development and even cause mortality ^[3][69]. The selection of appropriate media depends on several factors: type of basal media, strength of salts, vitamins, plant growth regulators, pH, gelling agents and other additives ^[19][100]. Due to the fact that the requirements of the nutrient composition vary by plant species and even cultivars ^[20][101], the medium often needs to be specifically optimised for optimum micropropagation performance measured in plant quality and multiplication rate. The nutrient composition may vary depend on the culture stage, such as induction, shoot regeneration, multiplication and rooting. In Duboisia micropropagation, limited studies are available on the testing suitability of basal nutrient formulations (Table 2). It is evident that all studies utilised MS medium except Luanratana ^[21][18], who used revised-tobacco medium for D. myoporoides and Yamada and Endo ^[22][27] who used B5 (Gamborg B5) medium ^[23][102] for D. leichhardtii F. Muell. There have been no studies conducted to compare the effect of basal nutrients on shoot regeneration, multiplication and rooting of this species. However, the effect of vitamins and salt strength on the culture development of Duboisia species has been discussed, but only confined to the callus growth stage. Lin ^[1][19] employed different strengths of MS salts (1/2, 1/4 and double strength) and various vitamins including MS vitamin, Griffin vitamin ^[24][88], Linsmaier and Skoog vitamin, and B5 vitamin to compare the callus growth of an M × L hybrid. Among various combinations of different MS salt strength and vitamins, the highest callus growth rate (0.164/tube/day) was obtained in the media containing full-strength MS salts and B5 vitamin. Likewise, Luanratana ^[21][18] applied 1/2, 1/4, 1/8 and full-strength MS salts into medium to compare the callus growth of M × L hybrid. The best callus growth was achieved on MS medium containing full-strength MS salts.

Plant growth regulators (PGRs) are chemical substances, either naturally occurring or synthetic, that influence the growth and development of plants by acting at cellular levels to elicit specific growth responses. PGRs play a crucial role in a wide range of physiological processes, such as cell division and elongation, organogenesis, and responses to biotic and abiotic stresses. The main plant hormones categorised under PGRs include auxins, gibberellins, cytokinins, ethylene and abscisic acid, and there have been other growth promotors and inhibitors/retardants discovered in recent years ^[28][103]. In tissue culture, incorporation of one or more PGRs in culture medium to ignite cellular responses to achieve shoot growth or root induction is a common practice ^[5][91]. The specific PGR for a specific function at optimum concentration is essential. Cytokinin as a PGR promotes cell division and stimulates axillary bud outgrowth, thus it is the main hormone used for shoot proliferation ^[29][104]. However, the plant physiology is complex and physiological changes are often governed by the interaction of more than one PGR. Hence, a low concentration of auxin and gibberellin are applied in conjugation with cytokinin to maintain the optimum plant health and vigour ^[30][31][72,105].

The reported work on Duboisia shoot regeneration highlights BA as the most frequently used cytokinin within a concentration range of 2–15 mgL⁻¹ (Table 3). Application of other cytokinins has also been evaluated; however, the two cytokinins kinetin and zeatin have been found to be more effective for callus induction ^[7][44]. The BA concentration of 3–4 mgL⁻¹ incorporated with 4 mgL⁻¹ NAA, or 1 mgL⁻¹ IAA has shown successful shoot induction of D. myoporoides ^[7][21][18,44]. The highest shoot induction frequency of 70% in M × L hybrid was obtained using dual cytokinins 5 mgL⁻¹ BA and 2.5 mgL⁻¹ 2-iP ^[1][19]. Most studies apply the same medium for both shoot induction and shoot multiplication phase, while a few authors have employed different medium compositions for shoot multiplication. Lin ^[1][19] subcultured shoot buds of M × L hybrid on MS medium containing 2 mgL⁻¹ kinetin and 0.1 mgL⁻¹ NAA for shoot multiplication. For D. myoporoides R. Br., Kukreja and Mathur ^[7][44] multiplied shoots by culturing the shoots on MS medium containing 3 mgL⁻¹ BA and 1 mgL⁻¹ IAA.

Root induction is one of the most critical and difficult steps in woody plant tissue culture; however, it may vary depending on the species or cultivar. According to Klerk et al. ^[32][106], major losses in plant micropropagation occur at the rooting stage due to poor and slow rooting. Several factors influence the root formation: shoot quality, nutrients, hormone composition and culture incubation condition. Good shoot quality supports root initiation and development ^[33][107]. Successful root induction requires optimum growth conditions and a balanced auxin–cytokinin interaction ^[34][108]. Therefore, during an in vitro rooting process, the regenerated shoots from a shoot regeneration medium are transferred into a root-promoting medium containing different PGRs with other suitable conditions for root induction and extension. Auxin is the primary PGR for root induction in plants ^[35][109]. There are different types of auxins available; indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and NAA are the most common auxins used in the horticulture industry. IBA and NAA are stable auxins compared to the naturally occurring IAA, which is prone to faster degradation upon radiation and high temperature ^[36][110]. Application of auxin in rooting media for continuous incubation is the most common method to induce roots during micropropagation ^[34][108].

Under in vitro conditions, Duboisia species have been shown to have limited rooting capacity without the application of exogenous auxin ^[7][44]. The literature supports IBA as the most commonly used PGR for the rooting of Duboisia species ^{[1][8][9][10][21][22][25][26]}[18,19,23,27,84,85,87,89]. Application of 4 mgL⁻¹ IBA in MS basal medium has resulted in a maximum 65% rooting for M × L hybrid and 68% for D. myoporoides ^[21][18]. However, the hormone requirements for the rooting of Duboisia species show a variance depending on the cultivar and culture types (Table 4). In rooting studies, NAA was reported to be superior to IBA and IAA for D. myoporoides R. Br. A maximum of 70–80% rooting has been achieved in static liquid MS medium containing 0.5 mgL⁻¹ NAA ^[7][44]. To date, rooting percentages have not exceeded 80% for any Duboisia species in the published literature.

^][112]. Cool white fluorescent light is frequently used for illumination ^[39][113] and the incubation temperature is maintained at 25 °C consistently, while a lower temperature (18 °C) is suggested for bulbous species and a higher temperature (28–29 °C) for tropical species ^[40][114]. For Duboisia species, the majority of studies reported incubation temperatures of 25 ± 2 °C with a photoperiod ranging from 12 to 16 h light using cool white fluorescent light and a corresponding dark period of 12 to 8 h respectively, or with a continuous dim light (Table 5).

] and Luanratana ^[21][18] are listed in Table 6. So far, the maximum acclimatisation survival reported in literature is 88% for D. myoporoides, and 68% for M × L hybrid by Luanratana ^[21][18].

Another essential factor for plant micropropagation is incubation conditions. Plant health in vitro is highly influenced by temperature and light regimes ^[37][111]. These conditions need to be set according to plant species and culture stages for micropropagation systems ^[38

Acclimatisation is a critical process which determines the success of any micropropagation process ^[42][116]. The in vitro regenerated plantlets have several structural differences due to long-term exposure to luxurious high humidity and low light conditions in vitro. In vitro plants have fewer stomates, lack a cuticle, possess non-functional roots, and have a poorly performing vascular system ^[43][117]. Therefore, direct transplantation would result in plant wilting, desiccation and susceptibility to bacterial and fungal infection ^[31][105]. To ensure the success of acclimatisation, great care is required to gradually train the in vitro generated plants to develop structural features to withstand dynamic external environment conditions.

For Duboisia species, studies on acclimatisation have been very limited, possibly due to the limited success in generating rooted plantlets through tissue culture. The acclimatisation protocols summarised from the work of Lin ^[1][19

Starting from explant sterilisation, there are several problems encountered in Duboisia tissue culture at every culture stage. Browning is one of the main problems of in vitro culture ^[21][18]. Duboisia is a woody species that produces polyphenol exudates under in vitro conditions, which can inhibit cell growth and cause mortality ^[44][45][118,119]. According to Luanratana ^[21][18], the reduction of salt strength in the medium can reduce browning. In addition, incorporation of ascorbic acid or activated charcoal at a concentration of 0.1% has been shown to reduce extra exudates. However, application of ascorbic acid has led to other complications, such as retarded growth.

Previous work conducted by Hiti Bandaralage et al. at the University of Queensland (unpublished data) has identified several challenges pertaining to Duboisia micropropagation. Vigorous callusing is one of the prominent problems in Duboisia micropropagation. Callus formation is characteristic of plants as a response to stress such as wounding and application of exogenous auxin and cytokinin ^[46][120]. Callogenesis is closely related to cell dedifferentiation and redifferentiation, which is important for organogenesis ^[47][121]. However, over callusing inhibits outgrowth of shoots/roots in plant tissue culture ^[46][120]. The balance of auxin to cytokinin ratio is essential for callus regulation, while silver nitrate is also effective to reduce callus formation in woody species ^[48][49][122,123]. Defoliation is another major problem for Duboisia micropropagation; the severity of this is especially high during continuous culture and root induction stage. Frequent subculturing is proven to be effective to overcome defoliation caused by continuous culturing. For auxin-induced rapid defoliation during the root induction period, Hiti-Bandaralage et al. ^[30][72] used silver nitrate or silver thiosulphate in the culture media for avocado. Thus, effect of silver nitrate and silver thiosulphate on auxin-induced defoliation is suggested to be evaluated for Duboisia species.

A commercially viable micropropagation system generally requires high rooting percentages and good functional roots to assist acclimatisation. In vitro root induction of Duboisia has been proven to be difficult and mostly produced non functional roots causing problems during acclimatisation stage. This could be the reason for lack of commercially viable tissue culture technology for the species.