The advancements made in tissue culture techniques has made it possible to regenerate various horticultural species in vitro as micropropagation protocols for commercial scale multiplication are available for a wide range of crops. Clonal propagation and preservation of elite genotypes, selected for their superior characteristics, require high degree of genetic uniformity amongst the regenerated plants. However, plant tissue culture may generate genetic variability, i.e., somaclonal variations as a result of gene mutation or changes in epigenetic marks. The occurrence of subtle somaclonal variation is a drawback for both in vitro cloning as well as germplasm preservation. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage. Several strategies have been followed to ascertain the genetic fidelity of the in vitro raised progenies comprising morpho-physiological, biochemical, cytological and DNA-based molecular markers approaches. Somaclonal variation can pose a serious problem in any micropropagation program, where it is highly desirable to produce true-to-type plant material. On the other hand, somaclonal variation has provided a new and alternative tool to the breeders for obtaining genetic variability relatively rapidly and without sophisticated technology in horticultural crops, which are either difficult to breed or have narrow genetic base. In the present paper, sources of variations induced during tissue culture cycle and strategies to ascertain and confirm genetic fidelity in a variety of in vitro raised plantlets and potential application of variants in horticultural crop improvement are reviewed.

/pdf/somaclonal-variations-and-their-applications-in-34x0z8l3la.pdf

Somaclonal variations and their applications in horticultural crops improvement

Summary Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were nonflowering. These two groups of plants produced 58%‐101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2011.00677.x

Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production.

Laboratory Protocols CIMMYT Applied Molecular Genetics Laboratory

Micropropagation of apple — A review

Summary Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway- compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpres- sion or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was function- ally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.

/pdf/gateway-compatible-vectors-for-high-throughput-gene-3o6tjm408q.pdf

Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species

Switchgrass is one of the most important biomass/bioenergy crops. For its improvement as a feedstock through biotechnological approach, we have developed a high throughput Agrobacterium-mediated transformation system for cv. Alamo and two new elite cultivars, Performer and Colony. Highly regenerable and transformation-competent embryogenic calli were identified and used for genetic transformation. GFP reporter gene was employed to identify transformation events at early stages and to guide modifications at various stages for improvement of transformation efficiency. The modifications included infection under vacuum, co-cultivation at desiccation conditions, resting between co-cultivation and selection, and supplement of L-proline in the callus culture and selection media. Transformation efficiency over 90% was routinely achieved for Performer, and around 50% for Alamo and Colony. The new system substantially improved switchgrass transformation efficiency and will significantly contribute to the genetic improvement of this important biofuel feedstock via biotechnological approach.

High throughput Agrobacterium-mediated switchgrass transformation

Sodium hydroxide pretreatment of genetically modified switchgrass for improved enzymatic release of sugars.

Somaclonal variation has been observed in many plant species and is an alternative way to create variants and expand the germplasm pool A large scale tissue culture experiment was conducted with St Augustinegrass, an important turfgrass species for the southern USA, to induce somaclonal variation to enlarge the germplasm pool for breeding efforts Using an improved protocol, approximately 7900 St Augustinegrass plants were regenerated from cv Raleigh, and 119 morphological variants were identified Among the variants, 115 had a semi-dwarf growth habit with shorter and narrower leaves, and shortened internodes and stolons However, 100 of them showed little vigour, which either grew very slowly or did not survive The remaining 15 showed reasonable growth vigour and were further investigated in the field Among them, 13 were semi-dwarf and 2 had longer leaves In addition, 2 other variants, with variegated (yellow striping) leaves, or significantly thicker stems were also observed and characterized The altered traits in the variant lines were stable during vegetative propagation and when grown in different environments

Tissue culture‐induced morphological somaclonal variation in St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

Dilute sulfuric acid pretreatment of transgenic switchgrass for sugar production.

St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is an important warm season turf and pasture grass. In vitro tissue culture of St Augustinegrass could serve as an important mean for its improvement through genetic transformation as well as induced somaclonal variation. To optimize tissue culture conditions for plant regeneration of St Augustinegrass, tissue culture responses of 11 explant tissues and four callus induction/subculture media have been examined. Embryogenic calli with regeneration potential were observed on cultures of early immature embryo [3 days after pollination (DAP)], immature embryo (7-14 DAP), and shoot base of young seedlings. The addition of benzyladenine (BA) in the callus induction/subculture medium enhances callus regeneration ability and does not harm callus induction for immature embryos. The best response came from 7 to 14 DAP immature embryo on MS medium containing 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l BA. The callus induction and regeneration rates were 97.7% and 47.6% respectively. However, BA supplement reduced callus formation and failed to enhance regeneration for young leaf bases. Scanning electron microscopy revealed that plant regeneration of St Augustinegrass is via somatic embryogenesis.

Ruyu Li

Papers

High throughput Agrobacterium-mediated switchgrass transformation

Sodium hydroxide pretreatment of genetically modified switchgrass for improved enzymatic release of sugars.

Tissue culture‐induced morphological somaclonal variation in St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

Dilute sulfuric acid pretreatment of transgenic switchgrass for sugar production.

Improved plant regeneration and in vitro somatic embryogenesis of St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]