Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates the cellular process of cryopreserved protocorm-like bodies (PLBs) was different comparative to non-cryopreserved PLBs. The cellular process was not only modified by the freezing and thawing effect but also due to the dehydration process itself during the cryopreservation procedure. Histological observation in Dendrobium Bobby Messina in encapsulation-dehydration method indicated that the degree of plasmolysis causes more cellular changes to the cryopreserved PLBs comparative to non-cryopreserved and stock culture PLBs. These results revealed higher amount of homogenous cell population and denser cytoplasm in cryopreserved PLBs. Histological analysis also revealed more voluminous nucleus in cryopreserved PLBs comparative to non-cryopreserved PLBs and PLBs stock culture. In contrast, scanning electron microscope analysis showed severe damages in cryopreserved PLBs and non-cryopreserved PLBs comparative to the PLBs stock culture which in return could be the possible reason of no regrowth in encapsulation-dehydration method. Damages incurred were on top part, side part, and at the stomata of the PLBs. Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates that the degree of plasmolysis causes changes in the cellular process of PLBs from cryopreserved PLBs was different comparative to non-cryopreserved PLBs.
Plant tissues such as somatic embryos, apical shoot tips, axillary shoot buds, embryogenic calli, and protocom-like bodies are potential micropropagules that have been considered for creating synthetic seeds. In the present study, 3-5 mm microshoots of Oryza sativa L. Cv. MRQ 74 were used as explant sources for obtaining synthetic seeds. Microshoots were induced from stem explants on Murashige and Skoog (MS) medium supplemented with 1.5 mg/L benzylaminopurine (BAP). They were encapsulated in 3% (w/v) sodium alginate, 3% sucrose, 0.1 mg/L BAP, and 0.1 mg/L α-Naphthalene acetic acid (NAA). Germination and plantlet regeneration of the encapsulated seeds were tested by culturing them on various germination media. The effect of storage period (15-30 days) was also investigated. The maximum germination and plantlet regeneration (100.0%) were recorded on MS media containing 3% sucrose and 0.8% agar with and without 0.1 mg/L BAP. However, a low germination rate (6.67%) was obtained using top soil as a sowing substrate. The germination rate of the encapsulated microshoots decreased from 93.33% to 3.33% after 30 days of storage at 4°C in the dark. Therefore, further research is being done to improve the germination rate of the synthetic seeds.
There is a pressing need for practical and successful conservation efforts to establish long-term germplasm collections of recalcitrant and tropical species, given the challenge and threat that these plants are facing. Cryopreservation is the only way of conserving some of these species, especially those with temperature or desiccation sensitive (recalcitrant) seeds. This review covers reports on cryopreservation studies of shoot tips (apical and axillary) of tropical and subtropical plants. Since many of these species have recalcitrant seeds, the cryopreservation successes, failures and problems involved with these seeds are also discussed. The methodologies, important factors and steps involved in successful cryopreservation protocols are analyzed. Finally strategies are suggested to develop a successful cryopreservation protocol for new plant species, in particular those with tropical recalcitrant seeds.
Seven different hormone treatments, namely 6-benzylaminopurine (BAP) at 2, 3 mg L(-1) was applied singly and in combination with Indole Acetic Acid (IAA) at 0.18, 0.8 and 1.8 mg L(-l), BAP at 3.3 mg L(-l) in combination with IAA at 1.8 and 3.3 mg L(-l) and triple combination of BAP at 2.3, IAA at 1.8 and Gibberellic acid (GA3) at 1.0 mg L(-1) were tested, over four different incubation periods of 30, 45, 60 and 75 days, for their effect in the proliferation and growth of Smooth cayenne pineapple shoot-tip culture. Combined application of BAP at 3.3 and IAA at 1.8 mg L(-1) induced the highest proliferation of 19 shoots/explant and the highest total of 121 and 125 shoots over 4 cycles of multiplication. Raising the IAA to 3.3 mg L(-1) resulted in the lowest proliferation and stunted shoots. Incorporation of GA3 improved the shoot length but caused drastic reduction in proliferation. The other treatments showed an intermediate effect.
Nowadays, many researches were conducted in minimizing tissue culture technology due to the overhead of cost needed. The purpose of this study was to investigate the effects of using five kinds of organic additives at four level concentrations responsive to the number of shoots produced for eight weeks in culture. Stem segment explants of Celosia sp. were cultured on MS medium that have been supplemented with different kinds of extract juice that serve as organic additives which are mature coconut, young coconut, papaya, banana and tomato at 20, 30, 50 and 70 ml L-1. The numbers of shoot on each explant were recorded and the mean of ten replicates explants were calculated. Among the media used, young coconut water at 70 ml L1- induced the highest shoot regeneration (14.21+/-8.26), followed by mature coconut water at 50 ml L-1 (13.14+/-10.33). Banana and tomato juice promote highest shoot regeneration of stem segments at 50 ml L-1 that produced 9.57+/-4.68 and 9.28+/-5.82 shoots per explants, respectively. While the lowest concentration which at 20 ml L-1 of papaya juice showed highest shoot regeneration (10.50+/-3.45) produced among the three other concentration tested. Statistical results showed that there were significant differences interactions effects (p<0.05) in terms of number of shoot regenerated between the types of extracts juices determined by ANOVA test. Comparing number of shoots regenerated that were cultured in control media, it showed higher than all of experimental medium composition. There were no big different in cost required in preparation of control media and the experimental media. Applications of five kinds of local fruit in tissue culture media should be considered since it responsive in shoot regeneration.
This paper reports the cryopreservation of Nephelium ramboutan-ake shoot tips derived from in vitro shoot multiplication and in vitro seed germination using vitrification. Preculture with either 0.5 M sucrose for 2 days or a combination of 0.3 M sucrose and 0.5 M glycerol for 3 days enhanced dehydration tolerance and resulted in the highest survival of shoot tips; however, none of the shoot tips withstood liquid nitrogen (LN) exposure. The use of a lower temperature (0 degree C) during exposure to plant vitrification solution (PVS2) led to higher survival of shoot tips, compared to exposure at 25 degree C. The survival percentage of shoot tips exposed to PVS2 for up to 20 min at 0°C was 83.3 percent. It was only 53.3 percent when shoot tips were exposed to PVS2 at 25 degree C for 5 min. The importance of vitamin C for reducing oxidative stress in shoots tips was demonstrated. The addition of 0.28 mM vitamin C during critical steps of the vitrification process resulted in a high survival (96.7 percent) without LN exposure, compared to 73.3 percent for shoot tips not treated with vitamin C. Moreover, 3.3 percent shoot tips withstood LN exposure when vitamin C was added during the loading step. This result suggests that cryopreservation is possible for this tropical, recalcitrant seeded tree species.
The use of artificial light sources such as light-emitting diodes (LEDs) has become a prerequisite in tissue culture studies to obtain morphogenetic enhancements on in vitro plants. This technology is essential for developmental enhancements in the growing plant cultures due to its light quality and intensity greatly influencing the in vitro growing explants at a cellular level. The current study investigates the effects of different light-emitting diode (LED) spectra on the growth of apical buds of Ficus carica var. Black Jack. Ficus carica, commonly known as figs is rich in vitamins, minerals, and phytochemicals capable of treating microbial infections and gastric, inflammatory, and cardiac disorders. Apical buds of Ficus carica var. Black Jack, presented morphogenetic changes when grown under six different LED spectra. The highest multiple shoots (1.80 per growing explant) and healthy growing cultures were observed under the blue + red LED spectrum. Wound-induced callus formation was observed on apical buds grown under green LED spectrum and discolouration of the growing shoots were observed on the cultures grown under far-red LED spectrum. Multiple shoots obtained from the blue + red LED treatment were rooted using 8 µM indole-3-acetic acid (IAA), and the rooted plantlets were successfully acclimatised. Compared with the other monochromatic LEDs, blue + red proved to be significantly better for producing excellent plant morphogeny. It is apparent that blue and red LED is the most suitable spectra for the healthy development of plants. The findings have confirmed that the combination of blue + red LED can potentially be used for enhancing growth yields of medicinally and commercially important plants.
Matched MeSH terms: Plant Shoots/growth & development
To explore the potential of in vitro rapid regeneration, three varieties (Golpaygan-181, Orumieh-1763, and Gorgan-1601) of sainfoin (Onobrychis viciifolia Scop. syn. Onobrychis sativa L.) were evaluated. For the first time, an encapsulation protocol was established from somatic embryogenic callus in torpedo and cotyledonary stages to create artificial seeds. Callus derived from different concentrations of Kinetin (0-2.0 mg L(-1)) and Indole-3-acetic acid (0-2.0 mg L(-1)) was coated with sodium alginate and subsequently cultured either in Murashige and Skoog (MS) medium or in soil substrate. Adventitious shoots from synthetic beads developed into rooting in full and half strength MS medium supplemented with various concentrations of auxin and cytokinin. Prolonged water conservation of black and red soils (1:1) had the highest rate of survival plantlets in the acclimatization process. Diverse resistance techniques in Onobrychis viciifolia were evaluated when the plants were subjected to water deficiency. Higher frequency of epicuticular waxes was observed in in vivo leaves compared to in vitro leaves. Jagged trichomes nonsecreting glands covered by spines were only observed in the lower leaf side. Ultimately, stomata indices were 0.127 (abaxial), 0.188 (adaxial) in in vivo and 0.121 (abaxial), 0.201 (adaxial) in in vitro leaves.
A procedure was developed for in vitro propagation of Curculigo latifolia through shoot tip culture. Direct regeneration and indirect scalp induction of Curculigo latifolia were obtained from shoot tip grown on MS medium supplemented with different concentrations and combinations of thidiazuron and indole-3-butyric acid. Maximum response for direct regeneration in terms of percentage of explants producing shoot, shoot number, and shoot length was obtained on MS medium supplemented with combination of thidiazuron (0.5 mg L(-1)) and indole-3-butyric acid (0.25 mg L(-1)) after both 10 and 14 weeks of cultures. Indole-3-butyric acid in combination with thidiazuron exhibited a synergistic effect on shoot regeneration. The shoot tips were able to induce maximum scalp from basal end of explants on the medium with 2 mg L(-1) thidiazuron. Cultures showed that shoot number, shoot length, and scalp size increased significantly after 14 weeks of culture. Transferring of the shoots onto the MS medium devoid of growth regulators resulted in the highest percentage of root induction and longer roots, while medium supplemented with 0.25 mg L(-1) IBA produced more numbers of roots.
The use of in vitro culture has been accepted as an efficient technique for clonal propagation of many woody plants. In the present research, we report the results of a number of experiments aimed at optimizing micropropagation protocol for tea (Camellia sinensis (L.) O. Kuntze) (clone Iran 100) using nodal segments as the explant. The effect of different combinations and concentrations of plant growth regulators (PGR) (BAP, TDZ, GA₃) on shoot multiplication and elongation was assessed. The influence of exposure to IBA in liquid form prior to transfer to solid media on rooting of tea microshoots was investigated. The results of this study showed that the best treatment for nodal segment multiplication in terms of the number of shoot per explant and shoot elongation was obtained using 3 mg/L BAP in combination with 0.5 mg/L GA₃. TDZ was found to be inappropriate for multiplication of tea clone Iran 100 as it resulted in hyperhydricity especially at concentrations higher than 0.05 mg/L. Healthy shoots treated with 300 mg/L IBA for 30 min followed by transfer to 1/2 strength MS medium devoid of PGR resulted in 72.3% of shoots producing roots and upon transferring them to acclimatization chamber 65% survival was obtained prior to field transfer.
This study focuses on the establishment of in vitro tuberization of Chlorophytum borivilianum using solid and liquid culture systems. A high in vitro tuberization rate on solid and stationary liquid Murashige and Skoog media was observed in the presence of 60 g l⁻¹ sucrose with 950, 1,265 and 1,580 µM 2-chloroethyl-trimethylammonium chloride (CCC). Application of a higher sucrose concentration of 90 g l⁻¹ showed a negative interaction with CCC on in vitro tuber number and days to in vitro tuber induction. For economic feasibility, 950 µM CCC with 60 g l⁻¹ sucrose was chosen as the best combination for in vitro tuberization in both solid and stationary liquid media. For optimization of in vitro tuber production,a comparison between solid, stationary liquid and shake liquid culture was carried out. Liquid culture with shaking at 80 r.p.m. resulted in a >2.5-fold increase in in vitro tuber production compared with solid culture.
Matched MeSH terms: Plant Shoots/drug effects; Plant Shoots/growth & development
The effect of preculture with different sugars and mannitol on cryopreservation of scalps of the banana (Musa) cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak was investigated. Scalps (0.3 square cm) were precultured on semi-solid MS-based medium, containing 0.4 or 0.5 M sucrose, glucose, fructose, trehalose or mannitol, for 14 days under a 16 h light and 8 h dark photoperiod prior to rapid cooling and storage in liquid nitrogen. Explants were rewarmed rapidly in a water bath at 40 degree C for 1 min, followed by recovery on two layers of sterile filter paper overlaying 25 ml aliquots of semi-solid MS-based medium with 5 mg per liter benzylaminopurine, 0.2 mg per liter indole acetic acid and 10 mg per liter ascorbic acid (PM8 medium) for 2 days in the dark. Subsequently, scalps were transferred onto 25 ml aliquots of semi-solid PM8 medium and incubated in the dark for 1 week prior to incubation in the light. Shoot regeneration from 5 - 48 percent of cryopreserved scalps of all the banana cvs., was observed only following preculture with 0.4 or 0.5 M glucose or fructose, and with 0.4 M trehalose for the cvs. Pisang Berangan and Pisang Awak. Preculture with 0.4 M glucose resulted in maximum shoot regeneration of cryopreserved scalps of 10 percent, 13 percent, 42 percent and 48 percent for the cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak, respectively. Concentrations of 0.5 M trehalose, or 0.4 and 0.5 M sucrose or mannitol were extremely toxic to scalps of all the cvs. investigated.
Matched MeSH terms: Plant Shoots/cytology; Plant Shoots/growth & development
Fresh water, coupled with soil salinization in many areas has resulted in an increased need forscreening of salt tolerant turf grasses. Relative salinity tolerance of eightwarm season turfgrass species were examined in this study in sand culture. Grasses were grown in a glasshouse, irrigated with either distilled water or saline sea water adjusted to 24, 48 or 72 dSm-1. Salt tolerances of the grasses were assessed on the basis of their shoot and root growth, leaf firing and turf quality. Regression analysis indicated that Zoysiajaponica (Japanese lawn grass) (JG), Stenotaphrum secundatum (St. Augustine) (SA), Cynodon dactylon (satiri) (BS), Zoysia teneuifolia (Korean grass) (KG), Digitaria didactyla (Serangoon grass) (SG), Cynodon dactylon (Tifdwarf) (TD), Paspalum notatum (Bahia grass) (BG) and Axonopus compressus(Pearl blue) (PB) suffered a 50% shoot growth reduction at 36.0, 31.8, 30.9, 28.4, 26.4, 25.7, 20.0 and 18.6 dSm1 of salinity, respectively and a root growth reduction at44.9, 43.7, 33.4, 31.0, 29.5 27.5, 21.5 and 21.4 dSm- of salinity, respectively. Leaf firing and turf quality of the selected species, as a whole, were also found to be affected harmoniously with the change in root and shoot growth. On the basis of the experimental results the selected species were ranked for salinity tolerance as JG>SA>BS>KG>SG >TD>BG>PB.
Matched MeSH terms: Plant Shoots/drug effects; Plant Shoots/growth & development
Allometry of shoot extension units (hereafter termed "current shoots") was analyzed in a Malaysian canopy species, Elateriospermum tapos Bl. (Euphorbiaceae). Changes in current shoot allometry with increasing tree height were related to growth and maintenance of tree crowns. Total biomass, biomass allocation ratio of non-photosynthetic to photosynthetic organs, and wood density of current shoots were unrelated to tree height. However, shoot structure changed with tree height. Compared with short trees, tall trees produced current shoots of the same mass but with thicker and shorter stems. Current shoots with thin and long stems enhanced height growth in short trees, whereas in tall trees, thick and short current shoots may reduce mechanical and hydraulic stresses. Furthermore, compared with short trees, tall trees produced current shoots with more leaves of lower dry mass, smaller area, and smaller specific leaf area (SLA). Short trees adapted to low light flux density by reducing mutual shading with large leaves having a large SLA. In contrast, tall trees reduced mutual shading within a shoot by producing more small leaves in distal than in proximal parts of the shoot stem. The production of a large number of small leaves promoted light penetration into the dense crowns of tall trees. All of these characteristics suggest that the change in current shoot structure with increasing tree height is adaptive in E. tapos, enabling short trees to maximize height growth and tall trees to maximize light capture.
The effects of sucrose preculture duration and loading treatment on tolerance of Garcinia cowa shoot tips to cryopreservation using the PVS2 vitrification solution were investigated. Ultrastructural changes in meristematic cells at the end of the preculture and loading steps were followed in an attempt to understand the effects of these treatments on structural changes in cell membranes and organelles. Increasing preculture duration on 0.3 M sucrose medium from 0 to 3 days enhanced tolerance to PVS2 solution from 5.6 percent (no preculture) to 49.2 percent (3-day preculture). However, no survival was observed after cryopreservation. Examination of meristematic cells by transmission electron microscopy revealed the progressive accumulation of an electron-dense substance in line with increasing exposure durations to 0.3 M sucrose preculture. Treatment with a loading solution (2 M glycerol + 0.4 M sucrose) decreased tolerance of shoot tips to PVS2 vitrification solution and had a deleterious effect on the ultrastructure of G. cowa meristematic cells. This study suggests that G. cowa meristematic cells may lose their structural integrity due to exposure to glycerol present in the loading solution at a 2 M concentration, either due to its high osmotic potential, or due to its cytotoxicity.
Aluminium (Al) phytotoxicity is an important soil constraint that limits crop yield. The objectives of this study were to investigate how growth, physiology, nutrient content and organic acid concentration is affected by Al, and to assess the degree of Al tolerance in different oil palm progeny (OPP). Four OPPs ['A' (Angola dura × Angola dura), 'B' (Nigerian dura × Nigerian dura), 'C' (Deli dura × AVROS pisifera) and 'D' (Deli dura × Dumpy AVROS pisifera)] were grown in different Al concentrations (0, 100 and 200 μm) in aerated Hoagland solution, pH 4.4, for 80 days. We observed a severe reduction (57.5%) in shoot dry weight, and root tips were reduced by 46.5% in 200 μm Al. In 'B' and 'C', the majority of macro- and micronutrients in plants were reduced significantly by 200 μm Al, with Mg being lowered by more than 50% in roots and shoots. The 200 μm Al treatment resulted in a 56.50% reduction in total leaf area, a 20% reduction in net photosynthesis and a 17% reduction in SPAD chlorophyll value in the third leaf. Root tips (0-5 mm) showed a significant increase in oxalic acid content with increasing Al concentration (∼ 5.86-fold); progeny 'A' had the highest concentration of oxalic acid. There was a significant interaction between Al concentration × OPP on total leaf number, root volume, lateral root length, Mg and K in root and shoot tissues, and Ca and N in shoots. The OPPs could be ranked in their tolerance to Al as: 'A' > 'D' > 'B' > 'C'.
Matched MeSH terms: Plant Shoots/drug effects; Plant Shoots/growth & development
Dendrobium sonia-28 is a popular orchid hybrid due to its flowering recurrence and dense inflorescences. Unfortunately, it is being decimated by fungal diseases, especially those caused by Fusarium proliferatum. In this study, selection of F. proliferatum-tolerant protocorm-like bodies (PLBs) was carried out by assessing the effects of differing concentrations of fusaric acid (FA). PLBs were cultured on Murashige and Skoog (MS) medium supplemented with 0.05 to 0.2 millimolar (mM) concentrations of FA. Higher concentrations of FA increased mortality of PLBs and reduced their growth. The survival rate for 0.05 mM FA was 20 % but only 1 % at the highest dose of 0.2 mM. Additionally, two different size ranges of PLBs were investigated, and growth increased more at lower FA concentrations for larger PLBs, whilst the growth rate of smaller PLBs was inhibited at an FA concentration of 0.2 mM. Histological examination using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses disclosed severe cell wall and organelle damage, as well as stomatal closure in PLBs treated with the high FA concentrations. Reductions in plantlet growth were much greater at the highest concentrations of FA. Some randomly amplified polymorphic DNA (RAPD) markers clearly discriminated between selected and non-selected variants of Dendrobium sonia-28, showing different banding patterns for each FA concentration and specific bands for selected and control plants.
The in vitro shoot proliferation of endemic Begonia pavonina in three culture conditions i.e semisolid medium (SM), liquid culture medium (LM) and in temporary immersion bioreactor system (RITA®) was analyzed in this study. To minimize contamination rates, seeds were surface sterilized and cultured on MS basal media. The clean raised shoots were then used as explants for inoculation onto the tested culture conditions. In this experiment, the explants were maintained in MS medium supplemented with 0.1mgL-1 BAP for shoot multiplication. After 4 weeks of incubation, higher regeneration rates were observed in TIM as compared to other medium conditions. The maximum shoot number was obtained from TIM system with a mean of 5.30 shoots per explant, followed by LM (2.47 shoots per explant) and SM (1.2 shoots per explant). Shoot hyperhydration was also lowest in a TIM system. Overall, TIM was shown to produce higher shoot multiplications combined with healthy morphological characteristics of plantlets. Shoot cultures from the all cultures were successfully rooted in vitro and acclimatized well in the greenhouse.
Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis.
A successful protocol was established for micropropagation in two selected varieties of exotic ornamental plants, Calathea crotalifera. The effects of different sterilization techniques, explant type, and the combination and concentration of plant growth regulators on shoots induction were studied. The axillary shoot buds explants sprouted from rhizomes in soil free conditions showed high induction rate of shoots with lowest contamination percentage when treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl2. In the present study, the highest number of multiple shoots was obtained in MS basal medium supplemented with 3.5 mg/L 6-Benzylaminopurine (BAP), 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 3% sucrose, and 6 g/L plant agar for both varieties and was used as multiplication medium. Microshoots were highly induced when the young shoot bud explants were incised longitudinally prior subculture. Chlorophyll analysis was studied to test the effects of activated charcoal and L-glutamine on reduction of necrosis problem. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L 1-Naphthaleneacetic acid (NAA) compared to indolebutyric acid (IBA). The complete regenerated plantlets were successfully acclimatized in the soilless medium under greenhouse condition. This is the first report of rapid mass propagation for C. crotalifera.