Chinnathambi Srinivasan

Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.)

Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of ectopic BBM expression on the development and regenerative capacity of tobacco (Nicotiana tabacum L.) through heterologous expression of Arabidopsis and B. napus BBM genes. 35S::BBM tobacco lines exhibited a number of the phenotypes previously observed in 35S::BBM Arabidopsis and B. napus transgenics, including callus formation, leaf rumpling, and sterility, but they did not undergo spontaneous somatic embryogenesis. 35S::BBM plants with severe ectopic expression phenotypes could not be assessed for enhanced regeneration at the seedling stage due to complete male and female sterility of the primary transformants, therefore fertile BBM ectopic expression lines with strong misexpression phenotypes were generated by expressing a steroid-inducible, post-translationally controlled BBM fusion protein (BBM:GR) under the control of a 35S promoter. These lines exhibited spontaneous shoot and root formation, while somatic embryogenesis could be induced from in-vitro germinated seedling hypocotyls cultured on media supplemented with cytokinin. Together these results suggest that ectopic BBM expression in transgenic tobacco also activates cell proliferation pathways, but differences exist between Arabidopsis/B. napus and N. tabacum with respect to their competence to respond to the BBM signalling molecule.

Plum (Prunus domestica) trees transformed with poplar FT1 result in altered architecture, dormancy requirement, and continuous flowering.

The Flowering Locus T1 (FT1) gene from Populus trichocarpa under the control of the 35S promoter was transformed into European plum (Prunus domestica L). Transgenic plants expressing higher levels of FT flowered and produced fruits in the greenhouse within 1 to 10 months. FT plums did not enter dormancy after cold or short day treatments yet field planted FT plums remained winter hardy down to at least −10°C. The plants also displayed pleiotropic phenotypes atypical for plum including shrub-type growth habit and panicle flower architecture. The flowering and fruiting phenotype was found to be continuous in the greenhouse but limited to spring and fall in the field. The pattern of flowering in the field correlated with lower daily temperatures. This apparent temperature effect was subsequently confirmed in growth chamber studies. The pleitropic phenotypes associated with FT1 expression in plum suggests a fundamental role of this gene in plant growth and development. This study demonstrates the potential for a single transgene event to markedly affect the vegetative and reproductive growth and development of an economically important temperate woody perennial crop. We suggest that FT1 may be a useful tool to modify temperate plants to changing climates and/or to adapt these crops to new growing areas.

Effect of nutrient media on axillary shoot proliferation and preconditioning for adventitious shoot regeneration of pears

The influence of the nutrient composition of plant tissue culture media on axillary shoot proliferation and their preconditioning effect on subsequent adventitious shoot regeneration from pear leaves was investigated. The goal was to improve both micropropagation and regeneration of ‘Bartlett’ and ‘Beurre Bosc’ pear cultivars. Driver–Kuniyuki walnut (DKW) and Quoirin and Lepoivre (QL) nutrient media were found to be superior to Murashige and Skoog (MS) and Woody Plant Medium (WPM) for axillary shoot proliferation. Shoots on WPM exhibited some chlorosis. Axillary shoot culture on DKW would be preferred to that on QL due to the production of excessively short thin shoots on the latter medium. DKW also was superior to QL and MS for production of young expanding leaves for use as explants in adventitious regeneration. Leaf explants derived from shoot proliferation cultures grown on DKW or QL media produced more adventitious shoots than leaf explants from MS.

Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

A brachytic dwarfism trait (dw) in peach trees is caused by a nonsense mutation within the gibberellic acid receptor PpeGID1c

Little is known about the genetic factors controlling tree size and shape. Here, we studied the genetic basis for a recessive brachytic dwarfism trait (dw) in peach (Prunus persica) that has little or no effect on fruit development. A sequencing-based mapping strategy positioned dw on the distal end of chromosome 6. Further sequence analysis and fine mapping identified a candidate gene for dw as a non-functional allele of the gibberellic acid receptor GID1c. Expression of the two GID1-like genes found in peach, PpeGID1c and PpeGID1b, was analyzed. GID1c was predominantly expressed in actively growing vegetative tissues, whereas GID1b was more highly expressed in reproductive tissues. Silencing of GID1c in plum via transgenic expression of a hairpin construct led to a dwarf phenotype similar to that of dw/dw peaches. In general, the degree of GID1c silencing corresponded to the degree of dwarfing. The results suggest that PpeGID1c serves a primary role in vegetative growth and elongation, whereas GID1b probably functions to regulate gibberellic acid perception in reproductive organs. Modification of GID1c expression could provide a rational approach to control tree size without impairing fruit development.

A minimal peach type II chlorophyll a/b-binding protein promoter retains tissue-specificity and light regulation in tomato

BackgroundPromoters with tissue-specificity are desirable to drive expression of transgenes in crops to avoid accumulation of foreign proteins in edible tissues/organs. Several photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues and would be good candidates for leaf and immature fruit tissue-specificity, if expression in the mature fruit were minimized.ResultsA minimal peach chlorophyll a/b-binding protein gene (Lhcb2*Pp1) promoter (Cab19) was isolated and fused to an uidA (β-glucuronidase [GUS]) gene containing the PIV2 intron. A control vector carrying an enhanced mas35S CaMV promoter fused to uidA was also constructed. Two different orientations of the Cab19::GUS fusion relative to the left T-DNA border of the binary vector were transformed into tomato. Ten independent regenerants of each construct and an untransformed control line were assessed both qualitatively and quantitatively for GUS expression in leaves, fruit and flowers, and quantitatively in roots.ConclusionThe minimal CAB19 promoter conferred GUS activity primarily in leaves and green fruit, as well as in response to light. GUS activity in the leaves of both Cab19 constructs averaged about 2/3 that observed with mas35S::GUS controls. Surprisingly, GUS activity in transgenic green fruit was considerably higher than leaves for all promoter constructs; however, in red, ripe fruit activities were much lower for the Cab19 promoter constructs than the mas35S::GUS. Although GUS activity was readily detectable in flowers and roots of mas35S::GUStransgenic plants, little activity was observed in plants carrying the Cab19 promoter constructs. In addition, the light-inducibility of the Cab19::GUS constructs indicated that all the requisite cis-elements for light responsiveness were contained on the Cab19 fragment. The minimal Cab19 promoter retains both tissue-specificity and light regulation and can be used to drive expression of foreign genes with minimal activity in mature, edible fruit.

Highly Efficient Transformation Protocol for Plum (Prunus domestica L.)

A high-throughput transformation system for plum has been developed using hypocotyl slices excised from zygotic embryos as the source of explants. The hypocotyl slices are infected in an Agrobacterium tumefaciens suspension and then cocultivated for 3 days in shoot regeneration ¾ MS basal medium supplemented with 9 μM 2,4-dichlorophenoxyacetic acid. Transgenic shoots are regenerated in a medium containing 7.5 μM thidiazuron and elongated in a medium containing 3 μM benzyladenine in the presence of 80 mg/L kanamycin in both media. Transformed shoots are rooted in ½ MS basal medium supplemented with 5 μM NAA and 40 mg/L kanamycin. The transgenic plants are acclimatized in a growth chamber and transferred to a temperature-controlled greenhouse. This protocol has allowed transformation efficiencies up to 42% and enabled the production of self-rooted transgenic plum plants within 6 months of transformation.

Loss of a highly conserved sterile alpha motif domain gene (WEEP) results in pendulous branch growth in peach trees

Significance Trees’ branches grow against the pull of gravity and toward light. Although gravity and light perception have been studied in model species, much is unknown about how trees detect and respond to these signals. Here, we report the identification of a gene (WEEP) that controls lateral branch orientations and is directly or indirectly required for gravity responses in trees. Loss or reduction of WEEP expression produced branches that grow outward and downward and did not exhibit normal gravitropism responses when displaced. WEEP is conserved throughout the plant kingdom and may be involved in gravity perception. WEEP may also be a valuable target for breeding or engineering trees with improved shapes for agricultural and landscaping applications. Plant shoots typically grow upward in opposition to the pull of gravity. However, exceptions exist throughout the plant kingdom. Most conspicuous are trees with weeping or pendulous branches. While such trees have long been cultivated and appreciated for their ornamental value, the molecular basis behind the weeping habit is not known. Here, we characterized a weeping tree phenotype in Prunus persica (peach) and identified the underlying genetic mutation using a genomic sequencing approach. Weeping peach tree shoots exhibited a downward elliptical growth pattern and did not exhibit an upward bending in response to 90° reorientation. The causative allele was found to be an uncharacterized gene, Ppa013325, having a 1.8-Kb deletion spanning the 5′ end. This gene, dubbed WEEP, was predominantly expressed in phloem tissues and encodes a highly conserved 129-amino acid protein containing a sterile alpha motif (SAM) domain. Silencing WEEP in the related tree species Prunus domestica (plum) resulted in more outward, downward, and wandering shoot orientations compared to standard trees, supporting a role for WEEP in directing lateral shoot growth in trees. This previously unknown regulator of branch orientation, which may also be a regulator of gravity perception or response, provides insights into our understanding of how tree branches grow in opposition to gravity and could serve as a critical target for manipulating tree architecture for improved tree shape in agricultural and horticulture applications.