Samir C. Debnath

An efficient in vitro shoot propagation of cranberry (Vaccinium macrocarpon ait.) by axillary bud proliferation

SummaryCultures of two eranberry (Vaccinium macrocarpon Ait.) cultivars, ‘Ben Lear’ and ‘Pilgrim’, and three eranberry clones from natural stands in Newfoundland were established in a nutrient medium containing N6[2-isopentenyl]adenine (2iP) from nodal and/or shoot-tip explants obtained under aseptic conditions. The cultivars differed in shoot regeneration in terms of shoot number per explant with various concentrations of 2iP over two culture periods. Best total shoot production was obtained when nodal segments were cultured in the medium supplemented with 2.5–5.0 mg 2iP l−1 (12.3–24.6 μM). With higher 2iP levels, shoots did not expand and had a high mortality rate. Nodal explants of the three clones cultured in the same nutrient medium supplemented with 2.5 mg 2iP l−1 (12.3 μM) produced three to five healthy axillary shoots per explant. In another experiment, nodal explants were more productive than shoot tips. In all experiments with subculture, there was an increase in shoot multiplication rate for all genotypes. Shoots were rooted in vitro in the same media used for shoot proliferation, but without any growth regulators. After their transfer to potting medium, almost all of the rooted plants survived. Cranberry genotypes can be efficiently propagated and maintained through nodal culture in a nutrient medium without auxin that contains 2.5–5 mg 2iP l−1 (12–25 μM).

In Vitro Culture of Lingonberry (Vaccinium vitis-idaea L.)

Abstract Shoots of three lingonberry (Vaccinium vitis-idaea L.) cultivars, ‘Regal’, ‘Splendor’, and ‘Erntedank’ and two clones from natural stands in Newfoundland were initiated in vitro from shoot tip and nodal explants on modified Murashige and Skoog (MS) medium containing the plant growth regulators N6-[2-isopentenyl]adenine (2iP) (12.3 μM) or zeatin (5.7 μM). Zeatin was more effective than 2iP, and induced proliferation of 2 to 3 times as many shoots in ‘Regal’ as 2iP. Out of four media tested for shoot proliferation, modified MS medium was found more effective than the Woody Plant Medium for shoot multiplication. With all three cultivars, a reasonable balance in terms of shoot multiplication rate and desirable growth characteristics was attained in a new medium. Nodal explants of the two clones cultured on the modified MS medium with 12.3 μM 2iP produced 4 to 6 healthy axillary shoots per explant in clone 1 and 2 to 4 shoots in clone 2. Shoots rooted well ex vitro directly on a 2 peat: 1 perlite (v/v) medium after dipping in 39.4 mM indole-3-butyric acid. The new protocol proposed from this study is expected to be adapted for propagating a wide range of lingonberry germplasm.

Strawberry sepal: Another explant for thidiazuron-induced adventitious shoot regeneration

SummaryAn efficient system to regenerate shoots on excised sepals (calyx) of greenhouse-grown ‘Bounty’ strawberry (Fragaria x ananassa Duch.) was developed in vitro. Sepal cultures produced multiple buds and shoots without an intermediary callus phase on 2–4 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ)-containing shoot induction medium within 4–5 wk of culture initiation. Young expanding sepals with the adaxial side touching the culture medium and maintained for 14 d in darkness produced the best results. In a second experiment, sepals proved more effective than the leaf discs and petiole segments for regenerating shoots. A third experiment compared the effects of six concentrations of two cytokinins (TDZ at 0, 0.5, 2, and 4 μM and zeatin at 2 and 4 μM) for elongation of sepal-derived adventitious shoots. The media containing TDZ generally promoted more callus formation and suppressed shoot elongation. TDZ-initiated cultures transferred into the medium containing 2–4 μM zeatin, produced usable shoots after one additional subculture. Shoots were rooted in vitro in the same medium used for shoot regeneration, but without any growth regulators. When transferred to potting medium, 85–90% of in vitro plantlets survived.

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.

Inter simple sequence repeat (ISSR) to assess genetic diversity within a collection of wild lingonberry (Vaccinium vitis-idaea L.) clones

Forty-three wild lingonberry [Vaccinium vitis-idaea ssp. minus (Lodd) Hult.] clones collected from four Canadian provinces were assessed for genetic variability by using inter simple sequence repeat (ISSR). Fifteen primers generated 356 polymorphic ISSR-PCR bands. A substantial degree of genetic diversity was found am ong the wild collections. Cluster analysis by the unweighted pair-group method with arithmetic averages (UPGMA) separated the wild clones into four main clusters, and identified the two remaining clones as outliers. Furthermore, within four clusters, the genotypes tended to form sub-clusters that were in agreement with the principal coordinate (PCO) analysis. Geographical distribution explained 10% of total variation as revealed by analysis of molecular variance (AMOVA). The ISSR markers detected a sufficient degree of polymorphism to differentiate among lingonberry clones, making this technology valuable for germplasm management and the more efficient choice of parents in current lingonberr...

Developing a scale-up system for the in vitro multiplication of thidiazuron-induced strawberry shoots using a bioreactor

The use of large-scale liquid cultures in a bioreactor system has the potential to resolve the manual handling of the various stages of micropropagation and increases shoot multiplication in vitro significantly compared with those cultured on semi-solid gelled medium. In an attempt to improve the micropropagation protocol for strawberry (Fragaria × ananassa Duch.), a procedure for the mass propagation of adventitious shoots regenerated from leaf, sepal and petiole explants of cultivar Bounty using a liquid medium-containing bioreactor system combined with gelled medium is described. Leaf disks, sepals and petiole halves produced multiple buds and shoots without an intermediary callus phase on 2-4 µM thidiazuron (TDZ)-containing shoot induction medium within 5-6 wk of culture initiation. TDZ supported rapid shoot proliferation at low concentrations (0.1 µM), but induced hyperhydricity in a bioreactor system. Bioreactor-multiplied hyperhydric shoots were transferred to gelled medium containing 2-4 µM zeatin...

Strategies to propagate Vaccinium nuclear stocks for the Canadian berry industry

Vacinium fruits are genetically heterozygous species characterized as “not coming true from seed”. Conventional methods for vegetative propagation of these species, although successful, are slow and labour-intensive, and few propagules can be produced from one plant of a selected clone or hybrid. Micropropagation techniques are important for clonal multiplication, germplasm im provement and gene conservation of Vaccinium fruits cultivated in Canada including blueberries, cranberries and lingonberries. In vitro propagation of these species using axillary bud proliferation and adventitious shoot regeneration has been investigated in a number of studies. Morphogenesis seems to be highly dependent on plant growth regulators and media used for culture, and this dependence is genotype specific. The paper presents the progress in-depth of various aspects of the in vitro culture of Canadian Vaccinium species for their commercial production. Also discussed are techniques for clone rejuvenation and plant tissue cul...

In Vitro Culture of Lowbush Blueberry (Vaccinium angustifolium Ait.)

SUMMARY Cultures of three lowbush blueberry (Vaccinium angustifolium Ait.) clones collected from natural stands in Newfoundland were established in vitro on a modified cranberry (V. macrocarpon Ait.) tissue culture medium containing zeatin (5 μM) or N6-[2-isopentenyl]adenine (2iP) (10 μM). Shoot proliferation with respect to shoot number per explant differed among clones at various concentrations of zeatin over two culture periods. Best total shoot proliferation was obtained when basal nodal segments were cultured in the medium supplemented with 2-4 μM zeatin. In another experiment, nodal explants were more productive than shoot tips. Shoots growing for more than 12 weeks on media that contained more than 4 μM zeatin occasionally produced adventitious shoot masses, which appeared to arise from dense calli growing at the base of the shoots in the medium. The lower concentration of sucrose and lower irradiance improved shoot proliferation with respect to vigor compared to the control treatments (30 g L_1 and 30 μmol m∼2 s_1, respectively). In all experiments with subculture, there was an increase in shoot multiplication rate for all clones. A 50-100-fold multiplication rate was obtained every 3 months with the clones.

Micropropagation of Small Fruits

The small fruit plants are predominantly woody perennial dicot angiosperms, bear small to moderate-sized fruits on herbs, vines, or shrubs; and are usually vegetatively propagated to maintain true-to-type. The importance of small fruits in horticulture lies in their dual role as in the landscape and of food. The fruits themselves are highly prized for their varying shapes, textures, flavors, and colors. Small fruits are highly nutritious and are used as snack and dessert foods and in beverages. The principal plant genera cultivated for their berry-like fruit areActinidia (kiwifruit or Chinese gooseberry) Fragaria (strawberries) Ribes (currants and gooseberries) Rubus (raspberries, blackberries, loganberries, etc.) Vaccinium (blueberries, bilberries, cranberries, lingonberries, whortleberries, etc.), and Vitis (grapes). Other small fruit species cultivated regionally include Amelanchier (June-or service berries or saskatoons) Sambucus (elderberries), and Viburnum (highbush cranberry or American cranberry bush).

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

Summary An efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.