Direct Plantlet Regeneration Research Articles

Effects of genotype and sodium p-nitrophenolate on microspore embryogenesis and plant regeneration in broccoli (Brassica oleracea L. var. italica)

Isolated microspore culture has been applied in breeding to obtain doubled haploids (DH) and therefore shorten the time of breeding process; however, the production of pure broccoli lines using DH produced in isolated microspore culture has not been sufficiently developed. This study aimed to establish an effective microspore culture protocol for broccoli for hybrid breeding purposes. Microspores of thirteen broccoli genotypes were successfully induced to form embryos in NLN-13 liquid medium. Three genotypes, BB1, BB4, and BB6, were cultured in NLN-13 medium containing sodium p-nitrophenolate (SPN) ranging from 0 to 0. 2 mg•L-1, to improve microspore embryogenesis and plantlet regeneration without callus development. The addition of 0.01–0.2 mg•L-1 SPN enhanced the frequency of microspore embryogenesis. Adding 0.1 mg•L-1 SPN yielded the maximum number of embryos per bud in BB1, BB4, and BB6, which were 1.92-, 2-, and 2.1-folds higher than the control, respectively. The suitable concentration (0.1 mg•L-1) of SPN application could enhance the frequency of direct plantlet regeneration for broccoli. Further, average spontaneous diploidization rate for BB1, BB4, and BB6 was 51.28%. This is the first study to utilize SPN in improving the frequency of microspore embryogenesis and plantlet regeneration, and offer an efficient method for the large production of DH lines that needed in hybrid seeds and genetic study in broccoli.

Development of an Efficient Micropropagation-based Agrobacterium-Mediated Genetic Transformation Protocol in Commercial Cultivar of Jute (Corchorus capsularis L.)

The present study describes an efficient in vitro culture protocol for direct plantlet regeneration and Agrobacterium- mediated genetic transformation of Corchorus capsularis L. cultivar JRC 517. In vitro morphogenetic capacity of different explants was used as parameter of performance adjudgement. Nodal explants with immature axillary buds when cultured on MS with 0.1 mg l-1 IAA and 0.1 mg l-1 Kin treatment showed maximum in vitro culture response (95%) with plantlets regeneration of 5.67 ± 0.30 %. A. tumefaciens strain LBA4404 harbouring a binary vector pBI121, containing gus A reporter gene under the transcriptional control of Cauliflower Mosaic Virus (CaMV) 35S promoter and NOS terminator was used in addition with neomycin phosphotransferase (npt-II) as plant selection marker gene. Different parameters were tested to accomplish utmost transformation competence, which indicates that O.D600. - of Agrobacterium cell suspension: 0.3; one day preculture; vacuum infiltration for 10 min at 600 mm Hg pressure; three days co-cultivation; 100 μM acetosyringone (AS) and 0.001 ml l-1 concentration of non-ionic surfactant (Tween 20) were found to be optimum treatment to achieve maximum number of stable genetic transformants (~3.6%). The putative transformants were screened on MS medium supplemented with 50 mg l-1 kanamycin (Kan50) and their transient expression was confirmed through GUS histochimical assay and PCR analysis. The survivor plants were grown under Kan 50 selection pressure, and rooted successfully. Regenerated plantlets were acclimatized, hardened and transplanted to green house. Stable integration of the transgene into the recipient genome was confirmed by PCR using compatible primers of gusA and nptII, and Southern hybridization. The transgenic plants showed normal morphology and most of them followed 3:1 ratio of Mendelian inheritance for single dominant locus. Our findings documenting the in vitro direct shoot regeneration protocol from nodal explants with concurrent transgenic development there from deemed to be successfully involving economically important gene/s and trait enrichment in jute in future.

Adventitious shoot induction from leaf segments in Anthurium andreanum is affected by age of explant, leaf orientation and plant growth regulator

Anthurium is one of the main potted plants on the flower market, and micropropagation is the most used technique for clonal and large scale propagation of this species. Calli, adventitious shoots and regenerated plantlets were obtained from leaf segments of Anthurium andraeanum. The influence of genotype, explant orientation, plant growth regulator, and age of donor plants were tested. We obtained regenerated plants in three (‘Red One’, ‘Red Dark’, and ‘Snow White’) out of four different cultivars. The ‘White Beauty’, however, was considered to be recalcitrant under these conditions. Therefore, the second study was conducted only with this recalcitrant cultivar, and the use of juvenile rather than adult leaf explants enhanced plantlet regeneration at the frequency of 0.3-2.8 plantlets per leaf segment. The use of 1.0 mg·L−1 6-benzylaminopurine (BAP) and no addition of 2,4-D in the culture medium resulted in direct plantlet regeneration of Anthurium. Plantlets obtained from ‘Red One’ and ‘White Beauty’ were successfully micropropagated and acclimatized. In ex vitro phases, leaf-derived plants of these two genotypes maintained their most important horticultural and ornamental characteristics.

Enhanced adventitious shoot regeneration from aerial stem explants of ginger using TDZ and its histological studies

A protocol for direct plantlet regeneration from aerial stem explants of ginger (Zingiber officinale Rosc.) has been developed. Adventitious shoots and roots were regenerated from in planta aerial stem explants of 2 ginger varieties viz. var. 'Jamaica' and var. 'Varada'. Maximum shoot and root regeneration were observed in the cultures containing TDZ and IBA (1:1; 1:0.1 mg L-1) in both varieties. Cultures containing TDZ alone showed poor shoot and root formation. Histological studies revealed that the shoot initials originated from the primary thickening meristem and apical meristem, while the root primordial originated from the primary thickening meristem present in the aerial stem. The hardening of in vitro plants was performed with different hardening media and successful acclimatization was obtained. The hardening medium containing soil: sand: coir dust: cow dung + 5 g Trichoderma harzianum / cup gave good result in all aspects like survival of hardened plants, plant height, number of leaves, and chlorophyll content.

Shoot and plantlet regeneration from meristems of Dioscorea rotundata Poir and Dioscorea alata L.

In vitro culture media capable of regenerating moderate to high shoots and/or plantlets from meristems of two yam species – Dioscorea rotundata and Dioscorea alata within comparable duration of 10 weeks as commonly obtained in other monocots and root and tuber crops were investigated. The study comprised 125 phytohormone combinations investigated in three factorial experiments each consisting of an auxin (NAA) and a cytokinin (BAP or kinetin), or two cytokinins only. The frequency of direct plantlet regeneration, though significantly (P and ranged from 0 to 10% at 3 weeks after culture (WAC) and 0 to 35% at 8 WAC. At 8 WAC, shoot regeneration of 42-75% was obtained in D. rotundata in MS medium supplemented with 0.1
M NAA + 0.20
M BAP, and shoot + plantlet regeneration of 60-82% obtained in media containing 0.05
M + 0.20
M BAP or 0.46
M BAP + 0.50
M kinetin in D. alata. Both shoot induction and plantlet regeneration were species-dependent. Induced shoots were successfully rooted in MS medium within 3 to 4 weeks, bringing time taken for plantlet regeneration to 11 to 12 weeks. Regenerants were morphologically similar to the mother plants. Results of the present study will facilitate regeneration of plantlets via meristem in D. rotundata and D. alata.

Effects of donor genotype and bud sampling on microspore culture of Brassica napus

The effects of genotype, intravarietal donor plants, planting density, inflorescence age, bud position, and bud developmental stage on embryo production in Brassica napus L. were investigated. Embryo yields, frequency of normal embryo production, and direct plantlet regeneration were genotype dependent. Embryo yield ranged from 4 to 196 embryos/anther equivalent, normal cotyledonous embryos varied from 6 to 72% of total embryos produced, and direct plantlet regeneration from embryos occurred from 1 to 47% of embryos subcultured. The G231 line was superior in all three factors Embryo production also was affected by the physiological condition of individual donor plants. Dense plantings reduced plant and bud size, leading to reduced microspore production. In some genotypes embryo production was affected negatively at dense planting densities (3 plants/pot), while in others the rate was unchanged, the plants maintaining high embryogenic potential. The highest frequency of embryogenesis was obtained from cultures of buds sampled at approximately 1 week after first flowering and decreased with the aging of the inflorescence. Buds from 2-week-old inflorescences produced fewer embryos and these were generally small, multilobed structures that never developed further into plants. Buds from the lateral racemes and from the primary raceme appeared to be equally capable of embryo production if selected at the appropriate stage of development. Embryogenic microspores commonly were found in buds with a petal/anther length ratio between 1/4 and 3/4, although the optimum embryo yield was from microspores of buds around the 1/2 ratio. Microspores from later bud stages became oval in culture and did not develop further, while uninucleate microspores remained round, and enlarged prior to division.