Development and evaluation of in vitro somaclonal variation in strawberry for improved horticultural traits

Strawberry cultivation is not popular in Bangladesh due to the unpredictable climatic conditions and lack of proper cultivars. Using somaclonal variation, several new promising selections were generated and evaluated for their flowering and fruiting ability, adaptability and sustainability. To induce variation, plants were regenerated using various tissue culture techniques. Our results suggested that a high concentration of BAP in culture medium successfully resulted in the induction of somaclonal variation. Among the tissue culture techniques adopted in this study, meristem culture was most effective for induction of somaclonal variation. Twenty five putative somaclones with better horticultural features were subsequently selected and field evaluated for three clonal generations. Several of the selections reverted back to their original phenotype within 2–3 vegetative propagations. Three of the stable selections were distinct from each other in terms of fruit and other horticultural characters, and have potential for commercial cultivation in Bangladesh.     

Effects of an in vitro maturation treatment on plant recovery from avocado zygotic embryos

An efficient protocol for in vitro maturation of very immature, <10 mm, avocado embryos has been developed. The efficiency of plant recovery as well as the quality of the resulting plants was greatly improved by including a maturation phase prior to induction of germination. The influence of different factors, such as the gelling agent, organic supplements or abscisic acid, on embryo maturation and subsequent germination was tested. Optimum conditions were met when maturation was carried out in B5m medium supplemented with the Jensen's amino acids, an extra 88 mM sucrose and 6 g l⁻¹ agar as gelling agent. At these conditions, embryos which had been collected 68 days after pollination germinated at a 65% rate in solid medium, giving rise to healthy and vigorous plantlets. Anatomical differentiation and storage product accumulation occurring during the in vitro maturation phase were studied by means of histological techniques. Results obtained revealed that, at the end of the in vitro maturation period, embryos resembled the pattern previously established for avocado embryos matured under in vivo conditions: histodifferentiation had been accomplished and starch granules and protein bodies were abundant.     

Micropropagation of Dendrobium draconis Rchb. f. from thin cross-section culture

An efficient procedure is outlined for rapid and mass in vitro propagation of an orchid, Dendrobium draconis Rchb. f. through in vitro culture of thin cross-sections (TCSs) derived from young stems. The TCS explants were excised along the stem from the base to shoot tip of 6-month-old plantlets and cultured on Murashige and Skoog (MS) medium supplemented with 20 g/l sucrose and different concentrations of N⁶-benzyladenine (BA), kinetin (Kn) and 1-naphthaleneacetic acid (NAA), either individually or in combination. Protocorm-like bodies (PLBs) were directly induced from the TCS explants and completely developed into shoots within 6–7 weeks. The optimal growth regulators combination for maximal PLB development was 2 mg/l BA and 1.0 mg/l NAA, giving rise to 68% of responding explants with an average 11 PLBs per explant. Shoot development was best achieved on MS medium containing sucrose and coconut water. Plantlets, 6–8 cm height were transplanted into coconut husk peat with 92% survival rate in a nursery.     

In vitro micropropagation of the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck and effects of sprayed GA3 after transplantation to ex vitro conditions

We established the conditions to micropropagate the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck through axillary shoot development from isolated areoles. For the shoot proliferation stage different explant orientation (vertical and horizontal), type of cytokinin (BA, DAP and K), and concentrations (0, 1.25, 2,5, 5.0 and 7.5 mg/L) were evaluated. Media [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Phys. Plant. 15, 473–497: 50 and 100%], and carbohydrate concentration (0.25, 0.5, 0.75 and 1.0%) were studied to optimize individual shoot growth and elongation. Following micropropagation and plantlet acclimatization, the effects of GA₃ on plant growth were determined by spraying a series of increasing concentrations (0, 150, 300 and 450 ppm). A reliable and efficient protocol of micropropagation was established for this particular plant species. The greatest propagation ratio (shoot proliferation) was obtained when explants were cultured in vertical orientation (4.975 shoots per explant) as compared to horizontal position (3.692 shoots per explant). The addition of BA to the media resulted in increased shoot number per explant (8) in comparison to K and DA, which produced only 2 shoots in average. However, after 42 days of culture, significantly higher shoot length was obtained with DAP (14 mm) compared to K and BA (4 mm). After the shoot proliferation stage, an elongation subculture was performed prior rooting in which shoot growth was enhanced when crowns of shoots were cultured in 50% of basal salt formulation of Murashige and Skoog (1962) and low sucrose concentration (2.5 and 5%). Exogenous application of GA₃ after plantlet acclimatization on glasshouse conditions increased spine-hair (developed from areoles in young plants) length as part of short-term effects. However, significantly higher values were obtained in plantlets treated with 300 ppm of GA₃ when compared with the rest of the treatments. At the end of the study, the most important long-term effect produced by GA₃ was the suppression of total shoot growth. The micropropagation protocol described here and the conditions to grow the plants through fertigation plus the application of GA₃ that induced changes in the phenotype may be used in commercial exploitations to regenerate 12,500 plantelts in average after 12 months of culture and produce healthy plants with better ornamental characteristics and higher commercial value.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.     

In vitro proliferation from axillary buds and ex vitro protocol for effective propagation of Syringa × hyacinthiflora ‘Luo Lan Zi’

As a precondition for lilac mass propagation, the optimal shoot-multiplication medium for Syringa × hyacinthiflora ‘Luo Lan Zi’ was ascertained mainly based on clustered microshoot inducement and large leaf area establishment in 6-benzyladenine (BAP) (1.00 mg L⁻¹) and zeatin (Z) (0.10 mg L⁻¹) combination. Medium supplied with lower level of BAP (0.50 mg L⁻¹) and auxin (IAA) (0.25 mg L⁻¹) was not suitable for lilac shoot proliferation, but it could be competent for long-term preservation of the un-rooted shoots so that subsequent proliferation culture could be carried out at anytime. In addition, excess height growth which resulted in low transplanting survival rate was effectively controlled by decrease in node number when paclobutrazol (PBZ) was applied in rooting medium at a concentration of 1.00 mg L⁻¹ after taking into account the effects on shoot height, rooting, persistent leaf area and PBZ carry-over. An important overwintering treatment was to use a plastic chamber covering for plants in the greenhouse prior to field planting to ensure adequate biomass of stem and underground parts not only in the current growing season but also in the subsequent years.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.