Vitro-grown Plantlets Research Articles

A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen.

Shoot tips of in vitro-grown plantlets of cassava (Manihot esculenta Crantz), representing a wide range of germplasm, were cryopreserved as follows: pre-cultured for 3 days, cryoprotected and dehydrated for 1 h, then frozen in liquid nitrogen using a six-step protocol. After 3 h in liquid nitrogen, the shoot tips were removed, rapidly warmed, and recultured sequentially in three recovery media. After 2 weeks, the regeneration of frozen shoot tips was completed. Genotypes with a low response were identified. Their response was attributed to the effects of pre and post-freezing steps. Refining the methodology led to a consistent 50-70% plant recovery.

Regeneration of Panax ginseng C.A. meyer by organogenesis and nuclear DNA analysis of regenerants

Plant regeneration ability of ginseng (t Panax ginseng C.A. Meyer) via organogenesis was studied. Compact callus was induced from four different types of explants-leaf, petiole, flower stalk, and root of t in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and kinetin (0.46 µM) were conditioned for two weeks on the same medium. These calli differentiated into adventitious shoots when cultured on 1/2MS basal medium plus kinetin 4.7 µM and silver thiosulphate 10 µM. An addition of GA3 (2.9 µM) and BA (4.4 µM) to MS basal medium, however, induced high frequency t in vitro flowering (86.1%) and multiple shoot budding which affected the normal complete development of plantlets. Plantlets with a well-developed root system were obtained six weeks after regenerated shoots had been transplanted to 1/2 MS20 medium containing IBA 1.2 µM. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all the regenerants were typically diploids as the mother plants were, indicating that nuclear DNA content remained stable during cell differentiation.

Developmental changes in carboxylase activities in in vitro cultured coconut zygotic embryos: comparison with corresponding activities in seedlings

Phosphoenolpyruvate Carboxylase (PEPC; EC: 4.1.1.31) and Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (RubisCO; EC: 4.1.1.39) enzyme specific activities were measured during the in vitro development of coconut (Cocos nucifera L.) zygotic mature embryos into plantlets and compared with those of palms produced by conventional seed germination. At the time of initiation of germination, high PEPC and low RubisCO activities were measured in both cultured and conventionally germinated embryos, thus indicating an anaplerotic CO2 fixation. During both in vitro and in planta development, RubisCO progressively took over and became the main route for inorganic carbon fixation. The in vitro-grown coconut plantlets showed a faster decrease in their PEPC:RubisCO ratio than the seedlings, suggesting that an earlier transition from a heterotrophic to an autotrophic mode of carbon fixation takes place in the in vitro-derived material. Just before acclimatization, the RubisCO activity in in vitro-derived plantlets (2.83 µmol CO2h−1mg−1 TSP) was lower than that in seedlings (6.98 µmol CO2h−1mg−1 TSP) of the same age. Nevertheless, after acclimatization, RubisCO activities were comparable in both in vitro and in planta germinated material

Regeneration of Panax ginseng C.A. Meyer by Organogenesis and Nuclear DNA Analysis of Regenerants

Plant regeneration ability of ginseng (Panax ginseng) via organogenesis was studied morphologically and anatomically. Compact callus was introduced from four different types of explants—leaf, petiole, flower stalk, and root—of in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (1.0 mg·L–1) and kinetin (0.1 mg·L–1) were conditioned for 2 weeks on the same medium. These calli differentiated into adventitious shoots when cultured on half-strength MS basal medium plus kinetin at 1.0 mg·L–1 and STS at 2.5 mg·L–1. An addition of GA3 (1.0 mg·L–1) and BA (1.0 mg·L–1) to MS basal medium, however, induced high-frequency in vitro flowering (86.1%) and multiple shoot budding, which affected the normal, complete development of plantlets. Plantlets with well-developed root systems were obtained 6 weeks after regenerated shoots had been transplanted to half-strength MS20 medium containing IBA at 0.25 mg·L–1. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all of the regenerants were typically diploids as were the mothers plants, indicating that nuclear DNA content remained stable during cell differentiation.

Cryopreservation Approach for the Germplasm Conservation of the Tropical Forest Tree Species

Cryopreservation approach for the germplasm conservation of three useful tropical forest tree species (Cedrela odorata L., Guazuma crinita Mart., and Jacaranda mimosaefolia D. Don.) were carried out using shoot-tip or roottip explants from in vitro-grown plantlets by four cryopreservation methods ((1) simple freezing, (2) rapid freezing, (3) slow pre-freezing, and (4) dehydration method). The effects of cold hardening treatments (5, 10, 15, and 20/10°C) and pre-culture (dehydration treatments) were also studied.The best results were achieved when the shoot-tips were cooled by slow pre-freezing before immersion in liquid nitrogen (LN). Survival and plant recovery rates of 50 and 20%; and, 50 and 15%; were obtained in cryopreserved shoot-tips of C. odorata and G. crinita, respectively. Although the effects of cold hardening and/or pre-culture treatments are not clear, apparently they were not effective in enhancing the survival or plant recovery rate after immersion in LN because these species may be intolerant of low temperature treatments of long duration and/or to drastic dehydration treatments.The cryopreservation of shoot-tips from tissue-cultured plants can be considered as a feasible alternative for the long-term storage of C. odorata, G. crinita, and J. mimosaefolia germplasm.

Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum

A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l-1 NAA, 0.1 mg l-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l-1 TDZ in combination with 0.1 mg l-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.

Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets

The number of chloroplasts per guard cell pair and the stomatal length of seventeen anther-derived plants obtained from three pepper donor genotypes were recorded. The haploid plants showed significantly fewer chloroplasts and shorter stomatal length than the diploid plants. Although both characters were positively correlated with the ploidy level, chloroplast number resulted a more reliable method compared to stomatal length to distinguish haploid from diploid in vitro-grown plantlets. The stomatal length values showed either significant variation among androgenic plants with the same ploidy or overlapping values between haploid and diploid individuals. On the contrary, the values for chloroplast number were more stable permitting discrimination of haploid from diploid plantlets regardless of their genetic background. Early ploidy estimation based on chloroplast counts of in vitro-grown plantlets may hasten pepper breeding programs utilizing anther-derived doubled haploids.

Water loss and polyethylene glycol-mediated acclimatization of in vitro-grown seedlings of 5 cultivars of date palm (Phoenix dactylifera L.) plantlets

Plantlets derived from shoot-tips of seedlings from five cultivars of date palm, Phoenix dactylifera L., were subjected to polyethylene glycol in liquid medium. Comparisons of water loss of detached leaves among in vitro-grown, polyethylene glycol-treated and greenhouse-grown plants showed significant differences with treatment for all cultivars studied. For each treatment, significant differences were also found among cultivars. The common result was that the percent of moisture loss of non-treated in vitro-grown plantlets was almost twice that of greenhouse-grown plants. Polyethylene glycol-treated plantlets showed a water loss of approximately 27%, similar to that of greenhouse plants as compared to an average of 40% in control plants. This demonstrates the possibility of using polyethylene glycol as an osmoticum for in vitro acclimatization of plantlets prior to transfer to soil.

In vitro acclimatization of date palm (Phoenix dactylifera L.) plantlets: A quantitative comparison of epicuticular leaf wax as a function of polyethylene glycol treatment

Wax deposits on leaf surfaces ofin vitro-grown plantlets,in vitro plantlets treated with polyethylene glycol and greenhouse-grown seedlings from five cultivars of date palm (Phoenix dactylifera L.) were extracted and quantified. Significant variations among treatments and cultivars were obtained. Greenhouse-grown plants had the greatest wax deposits followed by the acclimatized plantlets.In vitro plantlets had an average of 15% of the wax of greenhouse plants. Cultivar and plant age differences had a significant effect on the quantity of wax deposits. Greenhouse seedlings of 'Majhool', 'Deglet Noor' and 'Khadraoui' (cultivars grown under irrigation) had less wax accumulation than 'Zahidi' and 'Sayer', dryland cultivars.The increase in wax deposition as a result of polyethylene glycol treatment, explains in part, the decreased water loss observed in acclimatized plantlets when transferredex vitro.

Callus culture and plantlet production in Carica papaya (Var. Honey Dew)

In order to develop techniques for efficient callus production and regeneration in Carica papaya (Var. Honey Dew), lamina, petiole, stem and root explants from in vitro plantlets were cultured in media supplemented with 2.0 mg/1 IBA and 0.5 mg/1 BAP. Use of in vitro-grown plantlets as an explant source helped to avoid contamination common in papaya tissue culture. Callusing was maximum in root explants cultured in a modified MS (half-strength) medium. Shoot reganeration was maxium in root-derived callus grown in full-strength modified MS medium supplemented with 0.5 mg/1 IBA and 1 to 2 mg/1 kinetin. A histological study indicated that shoot buds originated from peripheral cell layers of the callus. Each shoot regenerated from callus was subcultured using a multiplication medium. Root formation was induced in all shoots treated in half-strength of modified MS medium containing 2 mg/1 IBA and rooted shoots were transferred successfully to the field.

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.

Cryopreservation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry

Alginate-coated shoot tips from in vitro-grown apple ( Malus domestica Borkh cv. Fuji) were successfully cryopreserved following dehydration. Shoot tips cold-hardened at 5°C for 3 weeks, were progressively precultured on MS agar media with increasing sucrose (0.1,0.4 and 0.7M) daily at 5°C. The precultured shoot tips trapped into alginate-coated beads containing 0.5 M sucrose were treated in a medium supplemented with 1.0 M sucrose for 16 h at 5°C. Beads containing 1 shoot tip were then dehydrated up to about 33% water content (fresh weight basis) on sterile dry silica gel at 25°C before being immersed in liquid nitrogen (LN). The average rate of shoot formation after warming was about 80%. This method was successfully applied to three apple, one mulberry and three pear species or cultivars. This encapsulation-dehydration method also permitted the shoot tips to be stored at -135°C for 5 months with little or no decrease in the rate of shoot formation. This modified method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of deciduous trees.

Comparison of Hydathode Structure in Micropropagated Plantlets and Greenhouse-grown ‘Queen Elizabeth’ Rose Plants

Abstract Foliar anatomical comparisons were made between in vitro-grown plantlets and greenhouse-grown plants of ‘Queen Elizabeth’ rose (Rosa sp.) using scanning and light microscopy. Each acuminate leaf apex and marginal serration had a terminal hydathode region composed of a glandular tip and a subterminal, adaxial group of sunken water pores. Leaf apices of greenhouse-grown plants had up to 35 water pores per hydathode, while cultured plantlets had < 20. Hydathodes of leaf serrations had up to 10 water pores in both sample groups. Water pores and stomata of plantlet leaves were open, while those of greenhouse-grown plants had smaller apertures or were completely closed. Internally, hydathodes were delimited by a bundle sheath extending below the vascular tissues and approaching the adaxial epidermis on each side of the water pore zone. Files of tracheary elements extended various distances into the leaf teeth. Small, irregularly shaped parenchyma cells (epithem) abutted on the xylem parenchyma cells, filling the space between the files of tracheary elements and the adaxial epidermis. The hydathodes of plantlet leaves were smaller with fewer water pores and reduced epithem than those of greenhouse-grown plants. Ex vitro guttation probably occurs as a result of increased water potential and high relative humidity when plantlets are transferred from culture to soil.

Hydathode Structure of Micropropagated Plantlets and Greenhouse-grown ‘Totem’ Strawberry Plants

Abstract Leaves of in vitro-grown plantlets and greenhouse-grown plants of ‘Totem’ strawberry (Fragaria × ananassa Duch.) were compared using scanning and light microscopy. Each apex and marginal serration of in vitro- and greenhouse-grown leaves had a terminal hydathode region. The leaf teeth were composed of an acuminate-mucronate tip, obscured in greenhouse-grown plants by an abaxial cluster of thick-walled unicellular trichomes, and a subterminal, adaxial group of sunken water pores. Water pores and stomata of plantlet leaves were open, whereas greenhouse-grown plant leaves had closed water pores and stomata or comparatively small apertures. Internally, the hydathodes of greenhouse-grown plants and cultured plantlets were delimited by a bundle sheath that extended below the vascular tissues, approaching the adaxial epidermis on each side of the zone of water pores. Between the epidermis and the vascular tissues were loosely arranged epithem cells. The hydathodes of plantlet leaves were smaller than greenhouse-grown plants, with fewer water pores and reduced epithem.

Hydathode Anatomy and Adaxial Water Loss in Micropropagated ‘Silvan’ Blackberry

Abstract Foliar anatomical comparisons were made between in vitro-grown plantlets and greenhouse-grown plants of ‘Silvan’ blackberry (Rubus sp.) using scanning and light microscopy. Each apex and marginal serration of in vitro- and greenhouse-grown leaves had a terminal hydathode region composed of a scattered, primarily adaxial, group of sunken water pores. Water pores and stomata of plantlet leaves were open, while greenhouse-grown plant leaves had closed water pores and stomata or comparatively small apertures. Internally, the hydathodes of both cultured plantlets and greenhouse-grown plants were delimited by a bundle sheath that flanked the vascular tissues and extended to the epidermis. Between the vascular tissues and the epidermis were loosely arranged epithem cells. The hydathodes of plantlet leaves were simpler than those of greenhouse-grown plants, with fewer water pores and reduced epithem. Water loss from detached leaves of plantlets occurred through both leaf surfaces, although more water was lost from the abaxial surface. In contrast, foliar water loss from severed leaf blades of greenhouse-grown plants was primarily abaxial.