Establishment of an in vitro plantlet regeneration protocol for unique varieties of brinjal (Solanum melongena L.) var. Mattu Gulla and Perampalli Gulla.

Brinjal (Solanum melongena L.) var. Mattu Gulla (MG) and var. Perampalli Gulla (PG) are unique varieties with distinct flavour cultivated in Udupi, Karnataka State, and are exposed to several biotic and abiotic stresses. An efficient and reproducible in vitro regeneration method is required to expedite the manipulation of these brinjal varieties to cope up with stress by tissue culture and gene transfer methods. The present study, reports a rapid and efficient in vitro regeneration protocol for these two varieties. The in vitro growth response was studied on Murashige and Skoog (MS) medium supplemented with 2, 4-D, BAP and IAA, and the plantlets were regenerated efficiently from callus cultures of leaf, cotyledon and hypocotyl explants. Among the three explants, the hypocotyl explants were found to have better callus induction and multiple shoot regeneration. High frequency of shoot initiation was achieved from hypocotyl derived calluses in MS media with 2.0 mg/L BAP and 0.5 mg/L IAA in MG and PG. Efficient and rapid shoot proliferation, and elongation were noted in MS medium with 1.0 mg/L BAP and 0.3 mg/L GA3. The in vitro regenerated shoots produced healthy roots when they were cultured on MS medium supplemented with 0.5 mg/L IBA. A significant difference was observed in percentage of callus induction, number of shoots per callus, shoot elongation and number of hardened plantlets of MG and PG. MG showed maximum response in all stages of culture than PG. Hardening of plantlets in tissue culture was achieved in three weeks. The hardened plantlets were grown in pots for further acclimatization in green house and finally transplanted to experimental garden where they developed into flowering plants and produced mature fruits with viable seeds.

Influência de diferentes meios de cultura sobre o crescimento de Pfaffia glomerata (Spreng. ) Pedersen (Amaranthaceae) para conservação in vitro / Influence of different culture media on the growth of Pfaffia glomerata (Spreng. ) Pendersen (Amaranthaceae) for conservation in vitro

Pfaffia glomerata ocorre em vários estados do Brasil e países limítrofes da região Sul às margens de rios e nas orlas das matas de galerias, é espécie hidrófita e heliófita. As raízes de espécies do gênero Pfaffia são usadas na medicina popular brasileira, especialmente como tônico, afrodisíaco e no controle do diabete. O objetivo deste trabalho foi estabelecer um banco de germoplasma in vitro de Pfaffia glomerata. O experimento em delineamento inteiramente casualizado foi conduzido com seis tratamentos: 1) MS + 2 por cento de sacarose + 4 por cento de sorbitol; 2) MS/2 + 2 por cento de sacarose + 4 por cento de sorbitol; 3) MS + 2 por cento de sacarose + 4 por cento de sorbitol + 2 mg L-1 de pantotenato de cálcio; 4) MS/2 + 2 por cento de sacarose + 4 por cento de sorbitol + 2 mg L-1de pantotenato de cálcio; 5) MS + 2 por cento de sacarose + 3 por cento de manitol + 2 mg L-1de pantotenato de cálcio; 6) MS/2 + 2 por cento de sacarose + 3 por cento de manitol + 2 mg L-1de pantotenato de cálcio. Os resultados obtidos foram submetidos à análise de variância e ao teste de separação de médias de Scott Knott. Os tratamentos um, três e quatro apresentaram, significativamente, o maior número de segmentos nodais por haste, quando comparados com os tratamentos dois, cinco e seis. O tratamento dois foi o mais indicado para a conservação in vitro da espécie por ter promovido menor crescimento das plantas (altura de 3,1±1,9 cm), alto índice de sobrevivência, 100 por cento de explantes com brotação e o maior número de brotos por explante, após seis meses de cultivo. Todas as plântulas produziram raízes e não houve formação de calos, também não ocorreu hiperhidricidade nos tratamentos avaliados. As plantas aclimatizadas apresentaram 100 por cento de sobrevivência no ambiente ex vitro. A manutenção de acessos de P. glomerata no banco de germoplasma in vitro é viável tanto do ponto de vista da conservação quanto economicamente.

Pfaffia glomerata occurs in several states of Brazil and its neighboring countries in the south region at riverbanks and gallery forests. It is a hydrophyte and heliophyte species. The roots of the genus Pfaffia are used in Brazilian folk medicine especially as tonic, aphrodisiac and to control diabetes. The aim of this work was to establish an in vitro germplasm bank for Pfaffia glomerata. The experiment was carried out in completely randomized design with six treatments: 1) DM + 2 percent sucrose + 4 percent sorbitol; 2) DM/2 + 2 percent sucrose + 4 percent sorbitol; 3) DM + 2 percent sucrose + 4 percent sorbitol + 2 mg L-1 calcium pantothenate; 4) DM/2 + 2 percent sucrose + 4 percent sorbitol + 2 mg L-1 calcium pantothenate; 5) DM + 2 percent sucrose + 3 percent mannitol + 2 mg L-1 calcium pantothenate; 6) DM/2 + 2 percent sucrose + 3 percent mannitol + 2 mg L-1 calcium pantothenate. Results were subjected to analysis of variance and Scott Knott test for mean grouping. Treatments 1, 3 and 4 had a significantly larger number of nodal segments per stem, compared to Treatments 2, 5 and 6. Treatment 2 was the most appropriate for the in vitro conservation of this species since it led to the lowest growth (3.1±1.9 cm height), high survival rate, 100 percent explants with sprouting, and the largest number of sprouts per explant after six months of culture. All seedlings produced root and showed no formation of calluses or hyperhydricidity under the evaluated treatments. Acclimatized plants showed 100 percent survival in the ex vitro environment. Maintaining P. glomerata accessions in an in vitro germplasm bank is viable both economically and for conservation.

Genetic stability in rice micropropagation

An efficient clonal propagation procedure for six rice varieties cultivated in Argentina was developed by using shoot tip cultures, and the genetic stability of the micropropagated plants was verified by isozyme analysis. One week old seedlings obtained on MS medium were sectioned and subcultured on MS medium (0.75% agar) supplemented with different combination and concentrations of cytokinins (BAP and KIN) and auxins (2,4-D and NAA). After four weeks of culture, multiple shoots were obtained. The best response was observed on MS supplemented with BAP 5 mg l(-1). Shoot clumps were multiplied in MS liquid medium containing BAP 5 mg l(-1). Profuse rooting was obtained after transfer to MS medium lacking growth regulators and with sucrose 8% (w/v). Complete plants were successfully transferred to soil and grown to maturity. ADH and EST patterns of micropropagated rice plants showed polymorphisms compared with plants of the original varieties. However, the zymograms of the seed derived progeny of the micropropagated plants were similar to that of the original varieties. These results indicate the maintenance of the genetic stability in the sexual progeny of micropropagated plants.

Somatic embryogenesis and plant regeneration in Gloriosa L.

Friable callus was initiated from shoot apices of Gloriosa superba L. on basal MS medium supplemented with 2, 4-D (4mg L(-1)) + Kn(5 mg L(-1)) + CH(10 mg L(-1)) + CW(20%). Subculture of callus on the same medium after 4-5 weeks showed induction of large number of somatic embryos, which was confirmed with histological studies. Development of embryoids in plantlet took place when the embryogenic callus was transferred to basal MS medium supplemented with BAP (5 mg L(-1)), CH(50 mg L(-1)) +CW(20%). Roots were developed by subculturing them on to the medium containing Kn or BAP (5 mg L(-1)) and IBA (4 mg L(-1)). Plantlets were successfully transferred to pots containing mixture of soil, sand and farmyard manure (2:1:1).