A. Ganapathi

Glutamine: A Suitable Nitrogen Source for Enhanced Shoot Multiplication in Cucumis sativus L.

Shoot tip explants of cucumber (Cucumis sativus L. cv. Poinsett 76) were cultured in vitro on Murashige-Skoog medium with L-glutamine, ammonium nitrate, adenine sulphate, asparagine, ammonium succinate, potassium nitrate and sodium nitrate as the nitrogen sources along with optimal concentration of 0.044 mM benzyladenine to study their effects on in vitro morphogenesis. The explants grown with 0.068 mM L-glutamine displayed the highest culture response (74.6 %) and greatest shoot number per explant (13.6) at the end of two subcultures. The explants cultured with other nitrogen sources resulted in low culture frequency and low number of shoots per explant accompanied by basal callusing and necrosis.

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 µM sucrose, 0.8 % agar, 3.62 µM 2,4-dichlorophenoxy acetic acid and 2.22 µM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant−1) was achieved on MS medium supplemented with 8.88 µM BA, 2.5 µM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 µM) favoured shoot elongation and indole 3-butyric acid (7.36 µM) induced rooting. Rooted plants were hardened and successfully established in soil.

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (Cucumis sativus L. cv. Poinsett76) via Agrobacterium tumefaciens.

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50xa0μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1xa0mgxa0L−1 benzyladenine (BA), 20xa0mgxa0L−1l-glutamine and 2xa0mgxa0L−1 phosphinothricin (PPT) or 100xa0mgxa0L−1 kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2xa0mgxa0L−1) containing MS media with 0.5xa0mgxa0L−1 gibberellic acid (GA3) and 0.6xa0mgxa0L−1 indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T0 and the progenies. T1 segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 µM BA and 1.59 µM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 µM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 µM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.

In Vitro Organogenesis

Legumes are important sources of proteins for the growing population in many developing countries of the world. Their production is limited due to the crop’s susceptibility to fungal, bacterial and viral diseases, insect pests and besides many other undesirable agronomic traits. Genetic improvement of legumes by classical breeding has met with limited success due to the lack of genetic variability within the germplasm. Strategies for increasing and stabilizing the production of legume crops depend on the development of varieties resistant to diseases, pests and with other desirable agronomic traits. Recent biotechnological advances have offered the opportunity to develop new germplasms. The development of such technology largely depends on the availability of efficient regeneration protocols. In the present review, regeneration via organogenesis in legumes is described. The advantages and limitations of this technique along with directions for future research are discussed.

Biochemical changes induced by accelerated ageing in Bambusa bambos seeds

Decrease in seed viability and germination rate may be caused by biochemical changes associated with seed ageing. Different biochemical assays were conducted to investigate the changes occurring at the ageing of Bambusa bambos seeds. A reduction in the total content of food reserves such as sugars, proteins and lipids were recorded. Decreased activity of peroxidase, acid phosphatase, alkaline phosphatase were also noticed during accelerated ageing. A substantial increase in total free amino acids and the activity of amylases confirms the degradation of stored biomolecules in seeds during ageing.

REGENERATION OF PLANTLETS FROM HYPOCOTYL DERIVED CALLUS OF MORUS ALBA

ABSTRACT Plantlets were regenerated from hypocotyl callus of Morus alba cv. MR2. Calli were established from hypocotyl segments on Murashige and Skoog (MS) medium supplemented with indoleacetic acid (0.5 mg/1) and benzyladenine (BA) (0.5 mg/1). They were transferred to MS medium with different concentrations of naphthaleneacetic acid NAA and BA for four weeks. Adventitious shoot buds were observed by transferring callus onto fresh Linsmaier and Skoog (LS) medium containing NAA (0.5 mg/1) and BA (0.75 mg/1). Shoots produced in vitro were rooted on MS medium with indolebutyric acid (0.75 mg/1).

SOMATIC EMBRYOGENESIS IN CELL SUSPENSION CULTURE OF COWPEA (VIGNA UNGUICULATA (L.) WALP)

ABSTRACT Embryogenic callus was induced from seedling leaf explants of cowpea (Vigna unguiculata (L.) Walp. cv. C152 on Murashige and Skoog (MS) medium containing 2.0 mg 1−1 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was noticed when this callus was transferred to MS liquid medium supplemented with 2 mg 1−1 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical or filamentous proembryos. Subsequent divisions in the proembryo led to globular, heart, torpedo-shaped, and cotyledonary-stage somatic embryos. Tiny plantlets were obtained by transferring the cotyledonary-stage somatic embryos to MS liquid medium containing 0.5 mg 1−1 2,4-D.

ISOLATION AND CHARACTERIZATION OF NACL RESISTANT CELL LINE OF MULBERRY

The selected NaCl tolerant clones ofMorus alba L. cv. MR2 grow better at higher concentration of NaCl than non-selected clones. With increasing NaCl concentration the Na+, Cl− and proline content increased more and K+ and Ca2+ content decrease less in selected clones in comparison with non-selected ones.