Duboisia is an Australian native woody species of the Solanaceae family, a crucial source of alka... more Duboisia is an Australian native woody species of the Solanaceae family, a crucial source of alkaloids, and is naturally extracted for pharmaceuticals. The alkaloid content of the four naturally occurring species of Duboisia, i.e., Duboisia myoporoides R. Br., Duboisia leichhardtii F. Muell., Duboisia hopwoodii F. Muell. and Duboisia arenitensis, is not conducive for large-scale commercial extraction. High-value hybrids between D. myoporoides R. Br. and D. leichhardtii F. Muell. have become the commercial crop for the industry. Propagation of these hybrids is key for progression of this industry, especially for the establishment and expansion of plantations and to replenish old plantations. Commercial propagation of Duboisia completely depends on cutting propagation to ensure true-to-type propagules. Cutting propagation of this species is associated with several challenges and has been a hurdle for industry expansion for many years. Micropropagation can be an efficient and sustainab...
Plants are constantly faced with biotic or abiotic stress, which affects their growth and develop... more Plants are constantly faced with biotic or abiotic stress, which affects their growth and development. Yield reduction due to biotic and abiotic stresses on economically important crop species causes substantial economic loss at a global level. Breeding for stress tolerance to create elite and superior genotypes has been a common practice for many decades, and plant tissue culture can be an efficient and cost-effective method. Tissue culture is a valuable tool to develop stress tolerance, screen stress tolerance, and elucidate physiological and biochemical changes during stress. In vitro selection carried out under controlled environment conditions in confined spaces is highly effective and cheaper to maintain. This review emphasizes the relevance of plant tissue culture for screening major abiotic stresses, drought, and salinity, and the development of disease resistance. Further emphasis is given to screening metal hyperaccumulators and transgenic technological applications for st...
Neptunia amplexicaulis is an herbaceous legume endemic to the Richmond area in central Queensland... more Neptunia amplexicaulis is an herbaceous legume endemic to the Richmond area in central Queensland, Australia and is one of the strongest known Selenium hyperaccumulators on earth, showing significant potential to be utilised in Se phytoextraction applications. Here a protocol was established for in vitro micropropagation of Se hyperaccumulator N. amplexicaulis using nodal segments from in vitro-germinated seedlings. Shoot multiplication was achieved on Murashige and Skoog (MS) basal media supplemented with various concentrations of 6-Benzylaminopurine (BA) (1.0, 2.0, 3.0 mg L−1) alone or in combination with low levels of Naphthaleneacetic acid (NAA) (0.1, 0.2, 0.3 mg L−1), with 2.0 mg L−1 BA + 0.2 mg L−1 NAA found to be most effective. Elongated shoots were rooted in vitro using NAA, with highest root induction rate of 30% observed at 0.2 mg L−1 NAA. About 95% of the in vitro rooted shoots survived acclimatization. Clonally propagated plantlets were dosed with selenate/selenite solu...
Improving clonal rootstock propagation of avocado has been a major industry/research challenge gl... more Improving clonal rootstock propagation of avocado has been a major industry/research challenge globally for many decades. Tissue culture has been a focus for clonal propagation with substantial advancements in recent years. In the process of tissue culture, avocado rootstocks display differences in rooting and acclimation capacity. Such differences may relate to the specific structural characteristics of the rootstock. This study aimed to investigate the structural difference during tissue culture in two rootstocks ‘Reed’ and ‘Velvick’, with differing rooting and acclimation capacity. Histological investigations were carried out of stem vasculature, leaves and roots of tissue cultured plantlets. Quantitative parameters; stomatal index, stomatal density, trichome density, vein-islet density and vein termination density were also analysed. Prominent fascicular cambium and fewer phloem fibres in stems positively correlated with rooting capacity. Acclimation success positively correlate...
Step Wise Protocols for Somatic Embryogenesis of Important Woody Plants, 2018
Avocado (Persea americana Mill.) is an ancient tree species belonging to the Order Ranales and Fa... more Avocado (Persea americana Mill.) is an ancient tree species belonging to the Order Ranales and Family Lauraceae. At present, the demand for avocado is growing rapidly due to its high nutritional value and reported health benefits. Much of this demand is centred on only a few cultivated varieties despite there being vast genetic diversity in the species. Preservation of germplasm is a proactive approach to address future breeding needs and to safeguard the diversity of cultivated crops including avocado. Present conservation strategies for Avocado (Persea spp.) solely rely on field collections, as seeds are highly heterozygous and recalcitrant to long-term storage. However, these field banks are constantly exposed to abiotic and biotic stresses. Moreover, the size of the gene pool, number of replications and quality of maintenance are restrained by the local environmental conditions, space and funding. In this chapter we present a protocol for cryopreservation of avocado somatic embryos as an alternative to field banks for long-term storage of germplasm. The protocol describes all stages of a vitrification-based cryopreservation and regeneration technique including induction, maintenance, maturation, cryo-treatment and germination of somatic embryos. This in vitro regeneration and storage system for avocado will impose low risk and is a space efficient conservation method that will be beneficial for future improvement of this invaluable horticultural crop.
Achieving sustainable cultivation of tropical fruits, 2019
Avocado is a high-value horticultural crop desired for its premium nutritional qualities. Commerc... more Avocado is a high-value horticultural crop desired for its premium nutritional qualities. Commercial trees are propagated by grafting wood from a fruiting cultivar onto a rootstock variety. Rootstocks are commonly seedlings derived from any available seed in the growing region. This often results in poor-quality rootstocks, further constrained by their genetic heterogeneity and seasonal availability. Clonal propagation of rootstocks developed through breeding or selection programs can improve productivity and allow orchard intensification due to their genetic homogeny. This chapter discusses advances in avocado tissue culture for clonal propagation. The chapter highlights the potential of this technology, as well as recent innovations in techniques such as micropropagation, meristem culture and micrografting, for improving the sustainable supply of high-quality avocado plants to support future avocado industry growth.
Capparis decidua is an underutilized plant with diverse applications in ethnomedicine and pharmac... more Capparis decidua is an underutilized plant with diverse applications in ethnomedicine and pharmacology, and also, the plant has the potential to be a source of functional food and nutrapharmaceuticals. Being grown in arid- and semi-arid environments, the crop is well adapted to adverse conditions, and offers an excellent system to study the adaptation of such species to harsh environments. Despite these merits, the biodiversity of C. decidua is challenged by several natural and man-made factors, and the species might be enlisted as an endangered plant if the situation prevails. In this context, developing a tissue culture protocol for mass clonal propagation gains importance, and in this purview, the present study was undertaken to optimize the micropropagation of C. decidua. The study investigated the impact of seasonal changes on the potential of explants and also chose the appropriate explant (shoot tips) for further experimentation. A total of twenty-five different media supplemented with various plant growth hormones and factors were assayed, out of which media MS-3 (1 mg/L BAP, 1 mg/L NAA; 15 mg/L adenine sulphate, and 0.2% activated charcoal) was found the most suitable for bud outbreak and shoot induction. The study also found that MS-22 with 2 mg/L silver nitrate used as an additive contributed to maximum biomass. Further, supplementing the media with adenine sulphate favoured shoot differentiation, while silver nitrate improves bud outbreak percentage. Also, the study identified SM-4 and RM-3 as the suitable media for shoot root multiplication, respectively. Altogether, the study has provided an optimal protocol for micropropagation of C. decidua that would further open the avenues for research in conservation as well as facilitates genetic and genomic studies in this important species.
Recent developments in the cryopreservation space has increased the trend in germplasm collection... more Recent developments in the cryopreservation space has increased the trend in germplasm collections established through cryopreserved in vitro material. Cryopreservation of recalcitrant seeds through embryos and embryonic axes, is not uncommon. Tropical and sub-tropical plants are not acclimated to the cold season, therefore have no in-built natural resilience to the cold. Also, larger seeds from trees, such as avocado (Persea americana Mill.), mango (Mangifera indica) and durian (Durio zibethinus L.) are sensitive to desiccation, chilling and freezing stress, making them unsuitable for seed banking or cryopreservation. Alternatively, as seeds do not carry the same genetic make-up as the mother plant, especially in the context of woody rainforest species of which the cross-pollination is dominant; seed conservation does not serve the purpose of germplasm preservation. Other plant material and methods are needed for these plants to be successfully stored in liquid nitrogen (LN). One s...
Plant Cell, Tissue and Organ Culture (PCTOC), 2020
Cryopreservation combined with in vitro culture offers a safe and cost-effective method to conser... more Cryopreservation combined with in vitro culture offers a safe and cost-effective method to conserve germplasm. Conservation of Persea spp. has been limited to heterozygous somatic embryos that are not true-to-type. A method for shoot-tip cryopreservation is vital to preserve the exact gene pool of interest. For the first time cryopreservation protocols for mature shoot tips of two avocado cultivars (cvs) ‘Velvick’ and ‘Reed’ , were established. In vitro shoots were subjected to two different optimised pre-treatments; (1) cv ‘Velvick’—high sucrose (0.3 M) or (2) cv ‘Reed’—low temperature (10 °C) incubation, over a 2-week period prior shoot tip dissection. Two different plant vitrification solutions, plant vitrification solution 2 (PVS2) and vitrification solution L (VSL) were tested at 0 °C for 0, 10, 20, 30 and 40 min. Vitrified shoots were evaluated for survival and regrowth at 2 and 8 weeks after vitrification treatment and either with or without liquid nitrogen exposure. The study revealed that the optimal exposure time for each cultivar varied with the cryoprotectant used. After liquid nitrogen cv ‘Velvick’ highest regrowth levels were observed with 20 min exposure to either PVS2 or VSL, however, vigorous plants were produced only from VSL treated shoots. In the case of cv ‘Reed’ highest regrowth levels were observed with 10 min exposure to PVS2 however only morphologically normal plants were recovered from VSL treated shoots. Cryopreservation of avocado shoot tips was successful using PVS2 and VSL with both recording similar recovery rates for ‘Velvick’ and ‘Reed’; although only vigorous and morphologically normal plants were developed from VSL treatments.
Avocado (Persea americana Mill.) is a tropical tree, of the Lauraceae family, genus Persea. On th... more Avocado (Persea americana Mill.) is a tropical tree, of the Lauraceae family, genus Persea. On the basis of the morphology of reproductive structures, this genus was divided into two subgenera, Eriodaphne and Persea (Koop, 1966); the subgenus Eriodaphne includes many species with small fruits and of no commercial importance, whereas the subgenus Persea includes only a small number of species
Improving avocado (Persea americana Mill.) rootstock propagation has been a major industry and re... more Improving avocado (Persea americana Mill.) rootstock propagation has been a major industry and research challenge globally for many decades. Avocado is an outcrossing species; thus, clonal propagation is the only way to propagate proprietary rootstocks. At present, avocado clonal propagation involves ex vitro rooting of tree cuttings at very poor efficiency, long turn-around time and high expense. A suitable mass clonal propagation process through in vitro culture technology could potentially revolutionize the avocado industry globally. Over the last four years, we have been developing an in vitro mass propagation technology for avocado. We observed avocado cultivars to differ in their ability to acclimatize. We hypothesized that this difference may relate to the specific structural characteristics of the cultivars. To test this, histological investigation of the stems, leaves, and roots of tissue cultured plants of two avocado cultivars; 'Reed' and 'Velvick' was carried out. Quantitative parameters; stomatal index, stomatal density, trichome density, vein-islet density, and vein termination density were analysed for comparison. Acclimatization success correlated to the presence of fully differentiated secondary xylem in the root suggested to maintain high water availability. Meanwhile, the presence of smaller epidermal cells, high stomatal density, less amount of xylem vessels in stem and reduced vein termination density correlated to reduced acclimatization success. These findings will succour optimization strategies for in vitro culture of difficult-to-acclimate cultivars of avocado but also, other woody perennials experiencing similar problems.
Duboisia is an Australian native woody species of the Solanaceae family, a crucial source of alka... more Duboisia is an Australian native woody species of the Solanaceae family, a crucial source of alkaloids, and is naturally extracted for pharmaceuticals. The alkaloid content of the four naturally occurring species of Duboisia, i.e., Duboisia myoporoides R. Br., Duboisia leichhardtii F. Muell., Duboisia hopwoodii F. Muell. and Duboisia arenitensis, is not conducive for large-scale commercial extraction. High-value hybrids between D. myoporoides R. Br. and D. leichhardtii F. Muell. have become the commercial crop for the industry. Propagation of these hybrids is key for progression of this industry, especially for the establishment and expansion of plantations and to replenish old plantations. Commercial propagation of Duboisia completely depends on cutting propagation to ensure true-to-type propagules. Cutting propagation of this species is associated with several challenges and has been a hurdle for industry expansion for many years. Micropropagation can be an efficient and sustainab...
Plants are constantly faced with biotic or abiotic stress, which affects their growth and develop... more Plants are constantly faced with biotic or abiotic stress, which affects their growth and development. Yield reduction due to biotic and abiotic stresses on economically important crop species causes substantial economic loss at a global level. Breeding for stress tolerance to create elite and superior genotypes has been a common practice for many decades, and plant tissue culture can be an efficient and cost-effective method. Tissue culture is a valuable tool to develop stress tolerance, screen stress tolerance, and elucidate physiological and biochemical changes during stress. In vitro selection carried out under controlled environment conditions in confined spaces is highly effective and cheaper to maintain. This review emphasizes the relevance of plant tissue culture for screening major abiotic stresses, drought, and salinity, and the development of disease resistance. Further emphasis is given to screening metal hyperaccumulators and transgenic technological applications for st...
Neptunia amplexicaulis is an herbaceous legume endemic to the Richmond area in central Queensland... more Neptunia amplexicaulis is an herbaceous legume endemic to the Richmond area in central Queensland, Australia and is one of the strongest known Selenium hyperaccumulators on earth, showing significant potential to be utilised in Se phytoextraction applications. Here a protocol was established for in vitro micropropagation of Se hyperaccumulator N. amplexicaulis using nodal segments from in vitro-germinated seedlings. Shoot multiplication was achieved on Murashige and Skoog (MS) basal media supplemented with various concentrations of 6-Benzylaminopurine (BA) (1.0, 2.0, 3.0 mg L−1) alone or in combination with low levels of Naphthaleneacetic acid (NAA) (0.1, 0.2, 0.3 mg L−1), with 2.0 mg L−1 BA + 0.2 mg L−1 NAA found to be most effective. Elongated shoots were rooted in vitro using NAA, with highest root induction rate of 30% observed at 0.2 mg L−1 NAA. About 95% of the in vitro rooted shoots survived acclimatization. Clonally propagated plantlets were dosed with selenate/selenite solu...
Improving clonal rootstock propagation of avocado has been a major industry/research challenge gl... more Improving clonal rootstock propagation of avocado has been a major industry/research challenge globally for many decades. Tissue culture has been a focus for clonal propagation with substantial advancements in recent years. In the process of tissue culture, avocado rootstocks display differences in rooting and acclimation capacity. Such differences may relate to the specific structural characteristics of the rootstock. This study aimed to investigate the structural difference during tissue culture in two rootstocks ‘Reed’ and ‘Velvick’, with differing rooting and acclimation capacity. Histological investigations were carried out of stem vasculature, leaves and roots of tissue cultured plantlets. Quantitative parameters; stomatal index, stomatal density, trichome density, vein-islet density and vein termination density were also analysed. Prominent fascicular cambium and fewer phloem fibres in stems positively correlated with rooting capacity. Acclimation success positively correlate...
Step Wise Protocols for Somatic Embryogenesis of Important Woody Plants, 2018
Avocado (Persea americana Mill.) is an ancient tree species belonging to the Order Ranales and Fa... more Avocado (Persea americana Mill.) is an ancient tree species belonging to the Order Ranales and Family Lauraceae. At present, the demand for avocado is growing rapidly due to its high nutritional value and reported health benefits. Much of this demand is centred on only a few cultivated varieties despite there being vast genetic diversity in the species. Preservation of germplasm is a proactive approach to address future breeding needs and to safeguard the diversity of cultivated crops including avocado. Present conservation strategies for Avocado (Persea spp.) solely rely on field collections, as seeds are highly heterozygous and recalcitrant to long-term storage. However, these field banks are constantly exposed to abiotic and biotic stresses. Moreover, the size of the gene pool, number of replications and quality of maintenance are restrained by the local environmental conditions, space and funding. In this chapter we present a protocol for cryopreservation of avocado somatic embryos as an alternative to field banks for long-term storage of germplasm. The protocol describes all stages of a vitrification-based cryopreservation and regeneration technique including induction, maintenance, maturation, cryo-treatment and germination of somatic embryos. This in vitro regeneration and storage system for avocado will impose low risk and is a space efficient conservation method that will be beneficial for future improvement of this invaluable horticultural crop.
Achieving sustainable cultivation of tropical fruits, 2019
Avocado is a high-value horticultural crop desired for its premium nutritional qualities. Commerc... more Avocado is a high-value horticultural crop desired for its premium nutritional qualities. Commercial trees are propagated by grafting wood from a fruiting cultivar onto a rootstock variety. Rootstocks are commonly seedlings derived from any available seed in the growing region. This often results in poor-quality rootstocks, further constrained by their genetic heterogeneity and seasonal availability. Clonal propagation of rootstocks developed through breeding or selection programs can improve productivity and allow orchard intensification due to their genetic homogeny. This chapter discusses advances in avocado tissue culture for clonal propagation. The chapter highlights the potential of this technology, as well as recent innovations in techniques such as micropropagation, meristem culture and micrografting, for improving the sustainable supply of high-quality avocado plants to support future avocado industry growth.
Capparis decidua is an underutilized plant with diverse applications in ethnomedicine and pharmac... more Capparis decidua is an underutilized plant with diverse applications in ethnomedicine and pharmacology, and also, the plant has the potential to be a source of functional food and nutrapharmaceuticals. Being grown in arid- and semi-arid environments, the crop is well adapted to adverse conditions, and offers an excellent system to study the adaptation of such species to harsh environments. Despite these merits, the biodiversity of C. decidua is challenged by several natural and man-made factors, and the species might be enlisted as an endangered plant if the situation prevails. In this context, developing a tissue culture protocol for mass clonal propagation gains importance, and in this purview, the present study was undertaken to optimize the micropropagation of C. decidua. The study investigated the impact of seasonal changes on the potential of explants and also chose the appropriate explant (shoot tips) for further experimentation. A total of twenty-five different media supplemented with various plant growth hormones and factors were assayed, out of which media MS-3 (1 mg/L BAP, 1 mg/L NAA; 15 mg/L adenine sulphate, and 0.2% activated charcoal) was found the most suitable for bud outbreak and shoot induction. The study also found that MS-22 with 2 mg/L silver nitrate used as an additive contributed to maximum biomass. Further, supplementing the media with adenine sulphate favoured shoot differentiation, while silver nitrate improves bud outbreak percentage. Also, the study identified SM-4 and RM-3 as the suitable media for shoot root multiplication, respectively. Altogether, the study has provided an optimal protocol for micropropagation of C. decidua that would further open the avenues for research in conservation as well as facilitates genetic and genomic studies in this important species.
Recent developments in the cryopreservation space has increased the trend in germplasm collection... more Recent developments in the cryopreservation space has increased the trend in germplasm collections established through cryopreserved in vitro material. Cryopreservation of recalcitrant seeds through embryos and embryonic axes, is not uncommon. Tropical and sub-tropical plants are not acclimated to the cold season, therefore have no in-built natural resilience to the cold. Also, larger seeds from trees, such as avocado (Persea americana Mill.), mango (Mangifera indica) and durian (Durio zibethinus L.) are sensitive to desiccation, chilling and freezing stress, making them unsuitable for seed banking or cryopreservation. Alternatively, as seeds do not carry the same genetic make-up as the mother plant, especially in the context of woody rainforest species of which the cross-pollination is dominant; seed conservation does not serve the purpose of germplasm preservation. Other plant material and methods are needed for these plants to be successfully stored in liquid nitrogen (LN). One s...
Plant Cell, Tissue and Organ Culture (PCTOC), 2020
Cryopreservation combined with in vitro culture offers a safe and cost-effective method to conser... more Cryopreservation combined with in vitro culture offers a safe and cost-effective method to conserve germplasm. Conservation of Persea spp. has been limited to heterozygous somatic embryos that are not true-to-type. A method for shoot-tip cryopreservation is vital to preserve the exact gene pool of interest. For the first time cryopreservation protocols for mature shoot tips of two avocado cultivars (cvs) ‘Velvick’ and ‘Reed’ , were established. In vitro shoots were subjected to two different optimised pre-treatments; (1) cv ‘Velvick’—high sucrose (0.3 M) or (2) cv ‘Reed’—low temperature (10 °C) incubation, over a 2-week period prior shoot tip dissection. Two different plant vitrification solutions, plant vitrification solution 2 (PVS2) and vitrification solution L (VSL) were tested at 0 °C for 0, 10, 20, 30 and 40 min. Vitrified shoots were evaluated for survival and regrowth at 2 and 8 weeks after vitrification treatment and either with or without liquid nitrogen exposure. The study revealed that the optimal exposure time for each cultivar varied with the cryoprotectant used. After liquid nitrogen cv ‘Velvick’ highest regrowth levels were observed with 20 min exposure to either PVS2 or VSL, however, vigorous plants were produced only from VSL treated shoots. In the case of cv ‘Reed’ highest regrowth levels were observed with 10 min exposure to PVS2 however only morphologically normal plants were recovered from VSL treated shoots. Cryopreservation of avocado shoot tips was successful using PVS2 and VSL with both recording similar recovery rates for ‘Velvick’ and ‘Reed’; although only vigorous and morphologically normal plants were developed from VSL treatments.
Avocado (Persea americana Mill.) is a tropical tree, of the Lauraceae family, genus Persea. On th... more Avocado (Persea americana Mill.) is a tropical tree, of the Lauraceae family, genus Persea. On the basis of the morphology of reproductive structures, this genus was divided into two subgenera, Eriodaphne and Persea (Koop, 1966); the subgenus Eriodaphne includes many species with small fruits and of no commercial importance, whereas the subgenus Persea includes only a small number of species
Improving avocado (Persea americana Mill.) rootstock propagation has been a major industry and re... more Improving avocado (Persea americana Mill.) rootstock propagation has been a major industry and research challenge globally for many decades. Avocado is an outcrossing species; thus, clonal propagation is the only way to propagate proprietary rootstocks. At present, avocado clonal propagation involves ex vitro rooting of tree cuttings at very poor efficiency, long turn-around time and high expense. A suitable mass clonal propagation process through in vitro culture technology could potentially revolutionize the avocado industry globally. Over the last four years, we have been developing an in vitro mass propagation technology for avocado. We observed avocado cultivars to differ in their ability to acclimatize. We hypothesized that this difference may relate to the specific structural characteristics of the cultivars. To test this, histological investigation of the stems, leaves, and roots of tissue cultured plants of two avocado cultivars; 'Reed' and 'Velvick' was carried out. Quantitative parameters; stomatal index, stomatal density, trichome density, vein-islet density, and vein termination density were analysed for comparison. Acclimatization success correlated to the presence of fully differentiated secondary xylem in the root suggested to maintain high water availability. Meanwhile, the presence of smaller epidermal cells, high stomatal density, less amount of xylem vessels in stem and reduced vein termination density correlated to reduced acclimatization success. These findings will succour optimization strategies for in vitro culture of difficult-to-acclimate cultivars of avocado but also, other woody perennials experiencing similar problems.
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