A common event during in vitro morphogenesis (either embryogenesis or shoot organogenesis) is the ability of somatic cells within the explants to de-differentiate and acquire “meristematic identity”. The developmental program of such meristematic cells can then be re-routed to form shoots or embryos depending on the imposed culture environment. The objective of this research is to investigate how the altered expression of Brassica genes regulating meristematic activity in vivo affects in vitro morphogenesis. It is predicted that ectopic expression of positive regulators of the shoot apical meristem, SHOOT MERISTEMLESS (STM) and ZWILLE (ZLL) which increase the pool of meristematic cells within the apical meristem, has a beneficial effect on somatic embryogenesis and shoot organogenesis. Conversely the over-expression of CLAVATA1 (CLV1), a negative regulator which depletes the pool of meristematic cells, should inhibit both processes. Over-expression of the Brassica STM in Arabidopsis enhanced the production of somatic embryos and shoots in vitro possibly by reducing the requirement of the tissue for exogenous auxin, which is the inductive signal for the production of embryogenic and organogenic cells. This was also accompanied by profound alterations in gene expression patterns affecting components of DNA methylation and glutathione metabolism, which are beneficial for embryo formation. The introduction of STM also enhanced Arabidopsis shoot organogenesis through profound transcriptional changes in cytokinin signalling. While the ectopic expression of the Brassica CLV1 inhibited both somatic embryogenesis and shoot organogenesis, the expression of ZLL had no effects on the production of somatic embryos but encouraged the formation of shoots. Taken together these results suggest the existence of similar genetic mechanisms regulating the formation of meristem cells in vivo and embryogenic/organogenic cells in vitro.

A common event during in vitro morphogenesis (either embryogenesis or shoot organogenesis) is the ability of somatic cells within the explants to de-differentiate and acquire “meristematic identity”. The developmental program of such meristematic cells can then be re-routed to form shoots or embryos depending on the imposed culture environment. The objective of this research is to investigate how the altered expression of Brassica genes regulating meristematic activity in vivo affects in vitro morphogenesis. It is predicted that ectopic expression of positive regulators of the shoot apical meristem, SHOOT MERISTEMLESS (STM) and ZWILLE (ZLL) which increase the pool of meristematic cells within the apical meristem, has a beneficial effect on somatic embryogenesis and shoot organogenesis. Conversely the over-expression of CLAVATA1 (CLV1), a negative regulator which depletes the pool of meristematic cells, should inhibit both processes. Over-expression of the Brassica STM in Arabidopsis enhanced the production of somatic embryos and shoots in vitro possibly by reducing the requirement of the tissue for exogenous auxin, which is the inductive signal for the production of embryogenic and organogenic cells. This was also accompanied by profound alterations in gene expression patterns affecting components of DNA methylation and glutathione metabolism, which are beneficial for embryo formation. The introduction of STM also enhanced Arabidopsis shoot organogenesis through profound transcriptional changes in cytokinin signalling. While the ectopic expression of the Brassica CLV1 inhibited both somatic embryogenesis and shoot organogenesis, the expression of ZLL had no effects on the production of somatic embryos but encouraged the formation of shoots. Taken together these results suggest the existence of similar genetic mechanisms regulating the formation of meristem cells in vivo and embryogenic/organogenic cells in vitro.

Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens infection. We propose that virus delivery of embryo-inducing genes is a better approach for transformation because A) inserts more than 800 nucleotides are unstable, and will spontaneously inactivate, B) virus DNA can migrate through plasmodesmata to cells around the infected cell, creating a gradient of embryonic potential, C) the virus DNA does not pass through the germ line and the seed will not contain virus. We propose this method of inducing embryogenesis will facilitate the stable transformation of cotton and will be beneficial to the cotton industry. Ectopic expression of AtBBM, AtSTM, and AtWUS GrWUS:meGFP from a constitutive CaMV 35S promoter produced plants with phenotypes similar to those described in previous studies overexpressing AtBBM, indicating that the AtBBM gene was functional. The cotton cotyledon infiltration of the pART27 constructs showed transformed cells in Coker 312 by GFP localization in the nucleus. Although GFP was detected, no visible embryos appeared from the cotyledon. Cotyledons infiltrated with Agrobacterium harboring overexpression vectors withered and aborted after ~2 weeks. The virus-based vector in tissue culture failed to increase transformation efficiency, resulting in no embryos. The combination of hormone concentration showed no contribution to increasing the transformation efficiency.