An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars

Abstract

BackgroundRice genome sequencing projects have generated remarkable amount of information about genes and genome architecture having tremendous potential to be utilized in both basic and applied research. Success in transgenics is paving the way for preparing a road map of functional genomics which is expected to correlate action of a gene to a trait in cellular and organismal context. However, the lack of a simple and efficient method for transformation and regeneration is a major constraint for such studies in this important cereal crop.ResultsIn the present study, we have developed an easy, rapid and highly efficient transformation and regeneration protocol using mature seeds as explants and found its successful applicability to a choice of elite indica rice genotypes. We have optimized various steps of transformation and standardized different components of the regeneration medium including growth hormones and the gelling agent. The modified regeneration medium triggers production of large number of shoots from smaller number of calli and promotes their faster growth, hence significantly advantageous over the existing protocols where the regeneration step requires maximum time. Using this protocol, significantly higher transformation efficiency (up to 46%) and regeneration frequency (up to 92% for the untransformed calli and 59% for the transformed calli) were achieved for the four tested cultivars. We have used this protocol to produce hundreds of independent transgenic lines of different indica rice genotypes. Upon maturity, these transgenic lines were fertile thereby indicating that faster regeneration during tissue culture did not affect their reproductive potential.ConclusionsThis speedy, yet less labor-intensive, protocol overcomes major limitations associated with genetic manipulation in rice. Moreover, our protocol uses mature seeds as the explant, which can easily be obtained in quantity throughout the year and kept viable for a long time. Such an easy, efficient and generalized protocol has the potential to be a major tool for crop improvement and gene-function studies on the model monocot plant rice.

Mature Embryo-Based in vitro Regeneration of Indica Rice Cultivars for High Frequency Plantlets Production

“…Genetic transformation of rice with Agrobacterium requires suitable regeneration system from a transformed callus and ironically, shoot regeneration represents a major bottleneck in this endeavour. Most of the indica rice genotypes, the world's most cultivated rice types, still remain less amenable to genetic transformation due to their poor regeneration potential (Sahoo et al, 2011) Although reporting is abundant on callus induction, regeneration and also transformation in japonica rice, it is limited in indica rice. In this context, the evaluation of new factors and their manipulation for efficient callusing and green plant regeneration from mature embryo in indica rice is still challenging (Niroula et al, 2005).…”

Agrobacterium-mediated genetic transformation of Fonio (Digitaria exilis (L.) Stapf)

“…In some cereals, hygromycin concentration of up to 100 mg/l has been used to select transgenic plants. In some rice genotypes, 50 mg/l hygromycin is sufficient to select transgenic tissues (Sahoo et al, 2011). Since the sensitivity of callus to hygromycin B was not checked, possibly the right concentration of the antibiotic was not used for the selection.…”

A NAP-Family Histone Chaperone Functions in Abiotic Stress Response and Adaptation

“…S3B). pCAMBIA1301-OsNAPL6 overexpression (Ox) and pFGC1008-OsNAPL6-knockdown (KD) constructs were then used for Agrobacterium tumefaciens-mediated transformation of rice (Sahoo et al, 2011) followed by molecular confirmation of the plants for their transgenic status (Supplemental Figs. S4 and S5).…”

“…Ox and KD plants were generated following standard procedures of cloning and plant transformation followed by selection, regeneration, hardening (Sahoo et al, 2011), and molecular confirmation of the transgenic status by various means as detailed in Supplemental Methods.…”