In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Buckwheat accumulates aluminum in leaves but not in seeds

Buckwheat (Fagopyrum esculentum Moench. cv Jianxi) is highly resistant to Al stress and is known to be an Al-accumulator. Pot experiments were carried out in a greenhouse to investigate the accumulation of Al in leaves and seeds of buckwheat. Plants were grown for 12 weeks in a strong acid soil amended with or without CaCO₃ at a rate of 1 g kg⁻¹ soil. Old leaves accumulated as much as 10 g kg⁻¹ Al of dry weight when the plants were grown in the acid soil, while the Al concentrations in leaves immediately adjacent to seeds, seed coats, and embryos were, on average, 4516, 41.2 and 7.7 mg kg⁻¹, respectively. The Al concentration significantly decreased in leaves when the plants were grown in the limed soil, and the Al concentrations in leaves immediately adjacent to seeds, seed coats, and embryos were, on average, 1586, 21.3 and 3.1 mg kg⁻¹, respectively. These results show that seeds accumulate much less Al than buckwheat leaves. The underlying mechanisms are discussed. Section Editor: H. Lambers     

Plant regeneration in <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> from cell suspension cultures

A method for plant regeneration in Robinia pseudoacacia L. from cell suspension culture was established. Non regenerative friable callus from hypocotyls and cotyledon explants from in vitro raised seedling induced on solid Murashige and Skoog (MS) medium supplemented with 0.05 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) was used for initiation of cell suspension cultures on same MS medium but without agar. Single cells were isolated after 3 d and the optimum cell density was 1–3 × 10⁴ cells per cm³ of the liquid MS medium. Plating efficiency was 29.6 % and callus formed within 4 weeks was subcultured and transferred to solid MS medium supplemented with 0.6 mg dm⁻³ benzyladenine (BA) along with 0.05 mg dm⁻³ α-naphthalene-1-acetic acid (NAA) for the induction of adventitious bud primordia. The shoots developed were isolated and re-cultured on MS medium containing 0.6 mg dm⁻³ BA. These microshoots after dipping in 1–2 cm³ of 10 mg dm⁻³ indole-3-butyric acid (IBA) for 24 h in dark were cultured on half strength solid MS medium supplemented with 0.05 % charcoal and showed 80–82 % rooting within 4 weeks.     

High efficiency organogenesis and analysis of genetic stability of the regenerants in <Emphasis Type="Italic">Solanum melongena</Emphasis>

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm⁻³ zeatin, 0.1 mg dm⁻³ indoleacetic acid and 10 g dm⁻³ sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0–2.0 mg dm⁻³ gibberellic acid and 4.0–8.0 mg dm⁻³ AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

Micropropagation of Sesbania rostrata from the Cotyledonary Node

Multiple shoots were induced from the cotyledonary nodes derived from seedling of Sesbania rostrata on Nitsch (1969; N) medium supplemented with various concentrations of benzyladenine (BA). 1 mg dm⁻³ BA proved to be the best, eliciting 5.8 ± 1.0 shoots per explant in 100 % cultures. The elongation of shoots was best at 2.0 mg dm⁻³ BA. The shoot proliferation capacity increased to 7.5 shoots per explant following transfer of explants to the fresh shoot multiplication medium (MS + 1.0 mg dm⁻³ BA), after an initial incubation of 30 d. To further enhance number of shoots per explant an alternative strategy of cultivation of mother explant on fresh shoot multiplication medium after excision of shoots was adopted. Following the repeated harvesting of shoots an average of 33 shoots per explant could be obtained. The in vitro regenerated shoots produced roots when transferred to half-strength MS medium supplemented with 3 % sucrose and 1 mg dm⁻³ IBA. The developed plantlets were planted in the soil and transferred to the field after an acclimatization period of 3 – 4 months. These plants produced flowers and fruits in the field and exhibited the development of prominent and more organized stem nodules as compared to the in vivo raised plants of the same age. This revised version was published online in July 2006 with corrections to the Cover Date.     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

Somatic embryogenesis and plant regeneration in Heracleum candicans Wall

A protocol has been developed for achieving somatic embryogenesis and plant regeneration from petiole-derived callus of Heracleum candicans Wall. Callus was initiated on MS medium supplemented with 0.5 mg l^–1 2,4-D and 0.5 mg l^–1 BAP and subcultured on a medium containing double strength MS macrosalts, 1 mg l^–12,4-D and 0.25 mg l^–1 Kn. Numerous globular embryos were formed on the surface of the callus upon transfer to auxin-rich MS medium that lacked cytokinins. The globular embryos differentiated into mature embryos only when 2,4-D was removed from the medium. Mature embryo formation was significantly influenced by the pH of the medium and the addition of AgNO₃ and ABA. Eighty-five percent of the somatic embryos were converted into plantlets when transferred to a medium supplemented with 0.01 mg l^–1 BAP and 0.01 mg l^–1 IBA. The regenerated plants have been established in soil and appear to be identical to the parent plants in morphology and chromosome number. Received: 5 November 1997 / Revision received: 9 February 1998 / Accepted: 19 February 1998     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.     

Efficient micropropagation of Robinia ambigua var. idahoensis (Idaho Locust) and detection of genomic variation by ISSR markers

Robinia ambigua var. idahoensis, presumably originated from interspecific hybridization of R. pseudoacacia L. and R. hispida L., is a multipurpose tree. Several reports have showed that in vitro micropropagation is a feasible method to produce large quantities of ‘clonal’ plants from R. pseudoacacia, however, no information is available on micropropagation of R. ambigua or the other assumed parental species, R. hispida. Here, we report on a tissue culture system for efficient micropropagation of R. ambigua plants by enhanced branching of axillary buds taken from a single branch of a donor tree. The culture system consists of sequential use of three media, namely, the bud-induction medium (MS medium supplemented with 0.8–1.4 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA), elongation medium (MS medium added with 0.35–0.5 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA) and root-induction medium (1/4 MS medium fortified with 1.7–2.5 mg l⁻¹ IAA and 0.1–0.5 mg l⁻¹ IBA). In addition, we investigated the genetic stability (relative to the donor plant) of a sample of 41 morphologically normal plants randomly taken from ca. 13,000 micropropagated plants, by using the inter-simple sequence repeat (ISSR) marker with 32 selected primers. We found that of the 226 reproducible bands scored, 24 were polymorphic (10.62%), thus pointing to the occurrence, though at a relatively low level compared with an earlier study on R. pseudoacacia, of genomic variation in these micropropagated plants. Further sequencing on seven loci underlying the variations showed that two had significant homology to known or predicted plant genes.     

Plant regeneration from shoot apical meristems of Melia azedarach L. (Meliaceae)

A protocol was developed for plant regeneration of Melia azedarach L. by in vitro culture of apical meristem (0.5 mm in length). The influence of six clones was investigated. The culture procedure comprised two sequential steps: 1) Induction of shoots by in vitro culture of axillary buds from adult trees (10–15 years old) by culture on Murashige and Skoog (1962) medium (MS) supplemented with 0.5 mg·dm⁻³ BAP (6-benzylaminopurine), 0.1 mg·dm⁻³ IBA (indolebutyric acid), and 0.1 mg·dm⁻³ GA₃ (gibberellic acid). The Multiplication of the regenerated shoots was achieved in MS + 0.5 mg·dm⁻³ BAP + 0.1 mg·dm⁻³ GA₃. 2) In vitro culture of the apical meristems from the regenerated shoots in MS medium (0.7 %) supplemented with various combinations of BAP and IBA. Maximum shoot proliferation was obtained on MS medium supplemented with 0.5 mg·dm⁻³ BAP and 0.1 mg·dm⁻³ IBA. Regenerated shoots were rooted on MS + 3.5 mg·dm⁻³ IBA (4 days) followed by subculture on MS lacking growth regulators (30 days). Complete plants were transferred to soil.     

A comparison of arsenic mobility in Phaseolus vulgaris, Mentha aquatica, and Pteris cretica rhizosphere

The ability of Phaseolus vulgaris, Mentha aquatica, and Pteris cretica to release arsenic (As) species from contaminated soil was tested in rhizobox experiments in three soils differing in their physicochemical parameters and total and mobile As concentration. Relatively low uptake of arsenic by P. vulgaris and M. aquatica resulted in very low and ambiguous changes in rhizosphere soil compared to bulk soil. However, there were observed differences in the distribution of the mobile As portion in soil to individual As species as affected by plant species and/or plantation conditions of these plants. Higher percentage of mobile arsenite in mint rhizosphere seems to be related to more reducing conditions during cultivation of these wetland plants. P. cretica planted in the soils containing between 36 and 1436 mg As kg⁻¹ was able to accumulate between 80 and 500 mg As kg⁻¹ in aboveground biomass. The extractable concentrations of As compounds in rhizosphere soil of P. cretica showed a clear depletion of arsenate (representing more than 90% of extractable arsenic) with the distance from plant roots. However, the As uptake mechanisms, as well as As transformation within hyperaccumulating fern plants, differ substantially from those in higher plants. Therefore the finding of suitable higher plant tolerant to the As soil contamination with good ability to accumulate As in aboveground biomass remains for the further research.     

Plant regeneration from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.)

A procedure is described to regenerate plants from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.). Somatic embryogenic calli were induced from ginger shoot tips on solid MS medium with half the concentration of NH₄NO₃ and supplemented with 1.0 mg l⁻¹ 2,4-Dichloroacetic acid (2, 4-D) and 0.2 mg l⁻¹ Kin. Rapid-growing and well-dispersed suspension cultures were established by subculturing the embryogenic calli in the same liquid medium. Protoplasts were isolated from embryogenic suspensions with an enzyme solution composed of 4.0 mg l⁻¹ cellulase, 1.0 mg l⁻¹ macerozyme, 0.1 mg l⁻¹ pectolyase, 11% mannitol, 0.5% CaCl₂ and 0.1% 2-(N-morpholino) ethane sulphonic acid (MES) for 12–14 h at 27°C with a yield of 6.27 × 10⁶ protoplasts g⁻¹ fresh weight. The protoplasts were cultured initially in liquid MS medium with 1.0 mg l⁻¹ 2, 4-D and 0.2 mg l⁻¹ Kin. Then the protoplast-derived calli (1.5 cm²) were transferred to a basal MS medium containing 0.2 mg l⁻¹ 2, 4-D, 5.0 mg l⁻¹ benzyladenine (BA), 3% sucrose and 0.7% agar. The white somatic embryos were transferred to MS medium lacking growth regulators for shoot development. Shoots developed into complete plantlets on a solid MS medium supplemented with 2.0 mg l⁻¹ BA and 0.6 mg l⁻¹ α-Naphthaleneacetic acid (NAA). In addition, the effects of AgNO₃, activated charcoal (AC) and ascorbic acid (AA) on browning of protoplast-derived calli are discussed.     

Direct shoot regeneration from immature inflorescence cultures of <Emphasis Type="Italic">Chlorophytum arundinaceum</Emphasis> and <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L⁻¹ BA, 150 mg L⁻¹ Ads, 0.1 mg L⁻¹ NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L⁻¹ IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.     

The effect of phenyl acetic acid on shoot bud induction, elongation and rooting of chickpea

A highly efficient protocol for plant regeneration from cotyledonary node of two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri used phenylacetic acid (PAA). The Murashige and Skoog (MS) medium supplemented with 2.0 mg dm⁻³ 6-benzylaminopurine (BAP) and 1.0 mg dm⁻³ PAA was used for induction of bud formation. Buds were elongated on MS medium supplemented either with only 0.75 mg dm⁻³ gibberellic acid (GA₃) or 0.2 mg dm⁻³ GA₃ + 0.6 mg dm⁻³ PAA. The elongated shoots were then transferred onto rooting medium containing 1 mg dm⁻³ PAA. The frequency of multiple shoot induction and rooting was higher in Annigeri as compared to ICCV-10. The complete plantlets with well-developed roots were transferred to pots containing sterilized soil and sand in the ratio 3:1 where they survived (74 %) and set normal seeds.     

Micropropagation of <Emphasis Type="Italic">Zingiber rubens</Emphasis> and assessment of genetic stability through RAPD and ISSR markers

Protocol was developed for high frequency in vitro multiplication of an endemic species, Zingiber rubens Roxb. The sprouted buds of the rhizomes were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 0.5–5.0 mg dm⁻³), indole-3-acetic acid (IAA; 0.5–2.0 mg dm⁻³), kinetin (KIN; 1.0–3.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0.5–1.0 mg dm⁻³) and adenine sulphate (ADS; 80–100 mg dm⁻³). MS basal medium supplemented with 3 mg dm⁻³ BA and 0.5 mg dm⁻³ IAA was optimum for shoot elongation. The elongated shoots (1–2 cm) were transferred to multiplication medium containing 2 mg dm⁻³ BA, 1 mg dm⁻³ IAA and 100 mg dm⁻³ ADS. The multiplication rate remained unchanged in subsequent subcultures. Upon ex vitro transfer, 85 % of plants survived. Genetic stability of micropropagated clones were periodically evaluated at an interval of 6 months up to 30 months in culture using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis and genetic uniformity in all regenerants was confirmed.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.     

An assessment of genetic fidelity of micropropagated plants of Chlorophytum borivilianum using RAPD markers

Rapid micropropagation was achieved in Chlorophytum borivilianum Santapau and Fernandes using shoot base as explants. Multiple shoots were induced on Murashige and Skoog’s (MS) medium supplemented with 3.0 mg dm⁻³ 6-benzylaminopurine, 0.1 mg dm⁻³ 1-naphthaleneacetic acid, 150 mg dm⁻³ adenine sulphates and 3 % saccharose. Rooting was readily achieved upon transferring the shoots onto half strength MS medium supplemented with 0.1 mg dm⁻³ indolebutyric acid and 2 % saccharose. Micropropagated plantlets were hardened in the greenhouse and successfully established in soil. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the micropropagated plants. Thirty one arbitrary decamers were used to amplify genomic DNA from in vitro and in vivo plant material to assess the genetic stability. All RAPD profile analysis from micropropagated plants was genetically similar to mother plants.