Cryopreservation of an endangered <Emphasis Type="Italic">Hladnikia pastinacifolia</Emphasis> Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.     

Cryopreservation of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l⁻¹ α-naphthalene acetic acid and 0.5 mg l⁻¹ 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m⁻² s⁻¹) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl₂ solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS₂) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.     

Cryopreservation of pharmaceutically important orchid Dendrobium chrysanthum Wall. ex Lindl. using vitrification based method

Our present study constitutes the successful and efficient protocol for cryopreservation of Dendrobium chrysanthum. D. chrysanthum Wall. ex Lindl. is a pharmaceutically valuable, ornamental epiphytic orchid of temperate and subtropical regions. On account of excellent herbal medicinal value and horticultural importance, D. chrysanthum is becoming rare due to over exploitation. For long-term conservation of this orchid, protocorm-like bodies of D. chrysanthum were excised and used for cryopreservation by encapsulation–vitrification. In this cryogenic procedure, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix (comprising of 3 % (w/v) sodium alginate and 0.1 M CaCl₂). Encapsulated protocorm-like bodies (PLBs) were then precultured on MS liquid medium supplemented with different concentrations of sucrose (0.06, 0.3, 0.5, 0.7 M), and loaded in a loading solution (comprised of 2 M glycerol and 0.4 M sucrose) for different duration to make the precultured PLBs tolerant to plant vitrification solution 2 (PVS2). Subsequently, the PLBs were subjected to PVS2 (Sakai et al. 1990) treatment at different time of exposure (minutes) and temperatures (0 °C and 25 °C). Encapsulated–vitrified PLBs were plunged directly into liquid nitrogen and stored for 1 h. Optimum result (survival 63.2 % and regrowth 59.9 %) was obtained when the beads treated with loading solution for 80 min followed by PVS2 treatment for 100 min. Regenerated plants showed normal morphology as that of control plants.     

Efficient cryopreservation of adventitious shoots of Begonia x erythrophylla using encapsulation–dehydration requires pretreatment with both ABA and proline

Despite the widespread use of tissue culture as a means of propagating begonias and concerns regarding the preservation of germplasm, little information is available on the cryopreservation of these commercially important plants. For this reason studies were conducted to develop an encapsulation–dehydration method for the cryopreservation of adventitious shoots of the rhizomatous begonia, Begonia x erythrophylla. Adventitious shoots of B. x erythrophylla were found to be sensitive to dehydration and very sensitive to freezing. While pre-treatment with 0.75 M sucrose significantly increased the percentage of encapsulated shoots surviving dehydration, pre-treatment with sucrose did not afford cryoprotection without prior dehydration. Addition of abscisic acid and proline to the pre-treatment medium significantly improved the percentage of shoots surviving freezing. Pre-treatment of shoots with a medium containing, 0.75 M sucrose, 3.8 μM abscisic acid and 2.15 mM proline resulted in greater than 50% of shoots surviving freezing.     

Cryopreservation of protocorm-like bodies (PLBs) of <Emphasis Type="Italic">Phalaenopsis bellina</Emphasis> (Rchb.f.) christenson by encapsulation-dehydration

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth.     

Multiplication and cryopreservation of vanilla (<Emphasis Type="Italic">Vanilla planifolia</Emphasis> ‘Andrews’)

A simple and efficient method for multiplication of vanilla (Vanilla planifolia) was developed using in vitro fragmented explants (IFEs) as propagules. IFEs were obtained after dissecting apices from in vitro propagated clusters of plantlets, by cutting the remaining base of these plant clusters into segments of about 1 cm in length. After 4 months of culture on multiplication medium, 100% of IFEs produced up to 15 new shoots per explant, providing an efficient additional method for in vitro propagation of vanilla that maximizes the use of available material. Cryopreservation of apices from in vitro grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%) of new shoots were obtained for apices derived from clusters of in vitro plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid MS medium with 0.3 M sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20–30 min; and exposure to plant vitrification solution PVS3 for 30 min at room temperature. Even though the cryogenic protocol needs to be optimized to improve results, this work represents the first successful report of cryopreservation of vanilla apices.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Advances in cryopreservation of vanilla (Vanilla planifolia Jacks.) shoot-tips: assessment of new biotechnological and cryogenic factors

The effect of dehydration, cryopreservation, and reculture conditions on growth recovery (%) of vanilla (Vanilla planifolia) shoot-tips was evaluated using a D-cryoplate procedure. Tissues were excised from in vitro grown plantlets, preconditioned on MS semisolid medium supplemented with 0.15 M trehalose for 1 d, loaded in a solution of 0.4 M sucrose or trehalose and 2 M glycerol for 30 min, and dehydrated within a laminar flow cabinet for various durations (30, 60, 90, 120, 150, and 180 min). The same preconditioning and loading treatments were compared using dehydration with vitrification solutions (PVS2 or PVS3) for 30 min at room temperature according to droplet-vitrification and V-cryoplate methods. The highest (33%) recovery of cryopreserved shoot-tips was achieved using the D-cryoplate method after 0.15 M trehalose preconditioning, loading with sucrose-glycerol solution and desiccation for 180 min. DSC analyses revealed that the osmotically active water (OAW) content of the shoot-tips was reduced from 77% (fresh weight basis) to 17% after the only effective drying duration (180 min). Melting endotherms indicated that crystallization events accompanied cryopreservation of the tissues. Proliferation of multiple shoots was obtained by indirect organogenesis. Histological analysis of the explants during post-cryopreservation recovery confirmed the organogenic nature of the callus formed after 3–4 mo of reculture in the dark on semisolid multiplication medium. This was followed by a secondary organogenesis on MS medium with kinetin (2 mg L⁻¹) and exposure to a photoperiod. At present, this is the most optimized cryopreservation protocol for shoot-tips of V. planifolia.

     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.     

Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration

The first efficient cryopreservation procedure for in vitro-grown shoot tips of raspberry (Rubus idaeus L.) has been developed based on encapsulation–vitrification (EnVi) and encapsulation–dehydration (EnDe). EnVi resulted in higher survival (85%) and regrowth (75%) of cryopreserved shoot tips than EnDe (65 and 50%, respectively). In both cryogenic procedures, shoots regenerated from cryopreserved shoot tips without intermediary callus formation. Histological studies showed that a much larger number of meristematic cells survived following EnVi than EnDe. The EnVi procedure was applied to seven raspberry genotypes with an average survival and regrowth of 71 and 68%, respectively. Regenerated plants showed normal morphology. Results here indicate EnVi as a simple and efficient method for long-term preservation of R. idaeus germplasm.     

Effects of different spectral lights on <Emphasis Type="Italic">Oncidium</Emphasis> PLBs induction,proliferation, and plant regeneration

The effects of different spectral light distribution on in vitro induction and proliferation of Oncidium protocorm-like bodies (PLBs) and subsequent growth of plantlets were investigated. Shoot tips (5 mm in length) of proliferating shoots of Oncidium “Gower Ramsey” were vertically incubated on 1/2 Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 6-benzyladenine (BA), and grown under either monochromatic red light-emitting diodes (LEDs) (RR), blue LEDs (BB), yellow LEDs (YY) or green LEDs (GG). Cultures grown under fluorescent lamps (FL) were used as control. Selected FL-induced PLBs were cut into 3- to 4-mm sections and incubated on MS medium supplemented with 1.0 mg l⁻¹ BA and 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), and grown under RR, BB, YY, GG, or FL. Moreover, FL-differented shoots (15 mm in length with two leaves) were incubated on 1/2 MS medium with 0.5 mg l⁻¹ NAA, and grown under either FL, RR, 10% blue + 90% red LEDs (1BR), 20% blue + 80% red LEDs (2BR), 30% blue + 70% red LEDs (3BR), BB, 80% red + 10% blue + 10% far-red LEDs (RBFr), or 80% red + 10% blue + 10% green LEDs (RBG). Overall, the red light spectrum enhanced induction, proliferation, and the carbohydrate contents of PLBs, as well as subsequent plantlet lengths, while the blue spectrum promoted differentiation, protein accumulation, and enzyme activities in PLBs, as well as pigment content accumulation in PLBs and developing plantlets. The combination of red and blue LEDs resulted in higher energy efficiency as well as dry weight and enzyme activities in these plantlets.     

Cryopreservation of in vitro-grown shoot-tips of <Emphasis Type="Italic">Rabdosia rubescens</Emphasis> by encapsulation-dehydration and evaluation of their genetic stability

Shoot-tips of Rabdosia rubescens, excised from in vitro-grown proliferating shoots that were cold-hardened at 5°C for 3 weeks, were encapsulated in alginate beads. Subsequently, these were precultured in a mixture of 0.4 M sucrose and 2 M glycerol for 1 h and then desiccated with silica-gel to about 21% water content prior to freezing in liquid nitrogen. After thawing, about 85% of cryopreserved shoot-tips grew into true-to-type shoots and with enhanced rooting capacity. Eight single-bud sibling lines were used to assess genetic stability of these encapsulated shoot-tips. When the relative DNA content was measured by flow cytometry (FCM), no changes were observed between controls and cryopreserved shoots. Using a sequence-related amplified polymorphism (SRAP) assay, it was observed that seven out of eight cryopreserved lines showed identical banding patterns; while the eighth line displayed an absent band, amounting to a low variance rate of 0.01%. These findings suggested that it was necessary to monitor the genetic stability of recovered cryopreserved R. rubescens shoots.     

The effects of various parameters during processing for cryopreservation on the ultrastructure and viability of recalcitrant zygotic embryos of <Emphasis Type="Italic">Amaryllis belladonna</Emphasis>

Cryostorage (usually in, or above liquid nitrogen) is presently the only option for long-term germplasm conservation of species producing recalcitrant (desiccation-sensitive) seeds. The present study investigated the ultrastructural responses of zygotic embryos excised from recalcitrant Amaryllis belladonna seeds to the sequential steps involved in cryopreservation. Flash-dried embryos, with and without prior sucrose (non-penetrating) or glycerol (penetrating) cryoprotection, were cooled rapidly or slowly, recovered in vitro and then assessed for ultrastructural and viability responses. Untreated embryos were 100% viable, the ultrastructure being indicative of their actively metabolic condition. Although nuclear morphology changed, viability was unaffected after exposure to either glycerol or sucrose, but mitochondrial ultrastructure suggested enhancement of metabolic activity particularly after sucrose treatment. When flash dried after sucrose cryoprotection, a significant increase in the degree of vacuolation, abnormal plastid ultrastructure and some wall abnormality accompanied a decline in survival to 70% and 60% at water contents > and <0.4 g g⁻¹, respectively. In contrast, glycerol cryoprotection, which promoted retention of generally normal ultrastructure and also counteracted any increase in the degree of vacuolation, was associated with 100% and 90% survival of embryos at the higher and lower water contents. After exposure to liquid nitrogen (LN), ultrastructural irregularities were minimal in rapidly cooled glycerol-cryoprotected embryos, at water content <0.4 g g⁻¹, which showed 70% survival after retrieval from cryogenic conditions. At the other extreme, no embryos survived LN exposure when sucrose cryoprotected. The study relates the cumulative effects of subcellular abnormality and declining viability, in relation to experimental parameters for cryopreservation.     

Simplified cryopreservation of sweet potato [<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.] by optimizing conditions for osmoprotection

Shoot tips of sweet potato were successfully cryopreserved using an encapsulation vitrification method. Encapsulated shoot tips were pre-incubated in liquid Murashige-Skoog medium containing 30 g/l sucrose for 24 h, then precultured in sucrose-enriched medium (0.3 M sucrose) for 16 h. Shoot tips were osmoprotected with a mixture of 2 M glycerol and 1.6 M sucrose for 3 h before being dehydrated with a highly concentrated vitrification solution (PVS2) for 1 h at 25 degrees C. The encapsulated and dehydrated shoot tips were transferred to a 2 ml cryotube, suspended in 0.5 ml PVS2, and plunged directly into liquid nitrogen. Rapidly warmed shoot tips developed normal shoots and roots in 21 days without any morphological abnormalities after plating on a recovery medium. High levels (average of about 80%) of shoot formation were obtained for three cultivars of sweet potato. This encapsulation vitrification method appears promising for cryopreservation of sweet potato germplasm.     

Successful cryopreservation of Quercus robur plumules

Successful cryopreservation of Q. robur germplasm as plumules (i.e. shoot apical meristems of embryos) is described in this paper. After excision from the recalcitrant seeds and preliminary storage in 0.5 M sucrose solution (18 h), the plumules were subjected to cryoprotection (in 0.75 M sucrose, followed by 1.0 M sucrose and 1.5 M glycerol solutions), and next to desiccation (over silica gel or in nitrogen gas) and cooling (in slush at –210°C or in vials filled with liquid nitrogen, LN, −196°C), and were then cryostored for 24 h. High percentage of survival was obtained after cryostorage (21–67%, depending on pretreatment, assessed in vitro by greening plumules that increased in size). Desiccation of plumules over silica gel resulted in significantly higher survival after cryopreservation (58%) in comparison with desiccation in nitrogen gas (29%), with regrowth (shoots with leaves) 5–18%. The extent of plumule desiccation was comparable in both methods, in which drying of plumules for 20 min decreased the water content to 0.5–0.6 g H₂O g⁻¹ dry weight before LN exposure. The type of LN exposure did not significantly influence plumule survival and regrowth after cryostorage. Plumules isolated from acorns of four provenances survived cryostorage after cryoprotection followed by desiccation over silica gel and direct cooling in vials with LN (survival 51–76%, regrowth 8–20%). Normal plants developed from the recovered shoots after rooting. The presented protocol for Q. robur plumule cryopreservation may offer a potential approach for establishing germplasm conservation in gene banks for Quercus species.     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

Regeneration of <Emphasis Type="Italic">Clivia miniata</Emphasis> and assessment of clonal fidelity of plantlets

An efficient in vitro micropropagation system for Clivia miniata Regel was developed using basal tissues of young petals and young ovaries as explants. For callus induction, explants were incubated on Murashige and Skoog (MS) medium containing either 2.22 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 4.44 μM BA, 5.37 μM α-naphthaleneacetic acid (NAA), and 9.05 μM 2,4-D. Moreover, callus was induced from young ovaries when these were incubated on MS medium containing 8.88 μM BA, 10.74 μM NAA, and 9.05 or 18.10 μM 2,4-D. Subsequently, callus was transferred to MS medium supplemented with kinetin (KT) and NAA for shoot organogenesis. Frequency of shoot regeneration from petal-derived callus was highest when callus was transferred to medium containing 2.69 μM NAA with either 9.29 or 13.94 μM KT. Shoot regeneration frequency from ovary-derived callus was highest when this callus was transferred to medium containing 9.29 μM KT and 10.74 μM NAA. Overall, different explant types exhibited different organogenic capacities wherein, young petals had higher shoot regeneration frequencies than young ovaries. The highest rooting frequency (98.25 ± 3.04%) was obtained when shoots were transferred to half-strength MS medium without plant growth regulators. Regenerated plantlets were transplanted to soil mix and acclimatized, yielding a 96.80% survival frequency. Only 0.6% of regenerated plantlets exhibited morphological changes. The diploid status (2n = 22) of regenerated plantlets was determined using chromosome counts of root-tips. Moreover, inter-simple sequence repeats were used to assess the genetic fidelity of regenerated plantlets. Overall, regenerated plants shared 90.5–100.0% genetic similarities with mother plants and 89.0–100.0% similarities with each other.     

Direct shoot regeneration from nodal explants of <Emphasis Type="Italic">Sida cordifolia</Emphasis> Linn

A method was developed to initiate multiple shoots from mature nodal explants of Sida cordifolia Linn. High frequency of regeneration was achieved on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 6-benzylaminopurine, 0.5 mg l⁻¹ α-naphthalene acidic acid, 1.0 mg l⁻¹ adenine sulfate, and 10% (v/v) coconut milk. Multiple shoots were initiated within 21 d and the above media was capable of inducing the formation of more than 20 shoots from each explant. Regenerated shoots were successfully rooted on half-strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid and 3% (w/v) sucrose. Rooted plantlets were established in soil. The regenerated plantlets showed no morphological differences from the parent material. This protocol could be useful for germplasm conservation, cultivation, and genetic improvement of S. cordifolia.     

In vitro plantlet regeneration system from rhizomes and mannose-binding lectin analysis of <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis> Hua.

Polygonatum cyrtonema Hua. lectins (PCLs) were extracted from plantlets regenerated from rhizome explants of P. cyrtonema. Rhizome explants demonstrated a high frequency of callus induction (72.5%) and adventitious shoots differentiation (83.7%) on Murashige Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 1.0 mg l⁻¹ 6-benzyladenine. The adventitious shoots could root readily on 1/2 MS medium + 0.5 mg l⁻¹ α-naphthaleneacetic acid and regenerate plantlets with a survival rate of 75.0%. Regenerated rhizomes were freeze-dried, macerated and prepared for total RNAs and proteins extraction. The PCL gene was cloned and its expression level was measured by RT-PCR. Western blot using a lectin-specific antibody revealed a similar amount in regenerated rhizomes compared to wild rhizomes, Furthermore, lectin derived from regenerated rhizomes retained its ability to haemagglutinate rabbit blood cells.