Cytokinins play an important role in regulating plant growth and development. The cytokinin gene, isopentenyl transferase (ipt), was placed under the control of the ACC oxidase promoter from the LEACO1 gene from Lycopersicon esculentum and introduced into Nicotiana tabacum (cv. Havana) and chrysanthemum (Dendranthema × grandiflorum `Iridon'). Transformants were confirmed by PCR reaction and Southern blot and analyzed for phenotypical changes under both greenhouse and growth chamber conditions. With both species, LEACO1-ipt transgenic plants displayed a wide range of vegetative and generative phenotypes. With plants growing in the vegetative state, some LEACO1-ipt transgenic lines appeared similar to the non-transgenic wild-type cultivars while other lines showed excessive lateral branch development and short internodes. With plants grown under generative conditions, several LEACO1-ipt transgenic lines showed a 2 to 10-fold increase in the number of flower buds relative to the wild-type cultivars. With chrysanthemum, dramatic increases in bud count were observed on transgenic lines that otherwise displayed a morphology similar to the non-transgenic lines. Analysis of ipt expression indicated a marked change in gene expression between the most extreme phenotypes observed in this study. LEACO1-ipt lines that express normal vegetative development but increased flower bud counts appear to have great potential for ornamental crop improvement.
Richard J. McAvoy*, Mariya V. Khodakovskaya, Hong Liu, and Yi Li
Mariya V. Khodakovskaya, Richard J. McAvoy*, Hao Wu, and Yi Li
It has been reported that constitutive expression of the fatty acid desaturase enzyme increased the trienoic fatty acid content of thylakoid membranes in transgenic tobacco, allowing the membranes to remain fluid under cold conditions. While increased cold tolerance resulted from this genetic modification, plants with a constitutively expressed desaturase enzyme would not be particularly well suited for growth under warm temperatures. To increase the ability of plants to tolerate prolonged cold-storage and still perform under greenhouse production conditions (25 °C), a unique cold-inducible genetic construct was cloned and tested. The FAD7 gene, which encodes an omega-3-fatty acid desaturase enzyme, was put under the control of a cold-inducible promoter (cor15a) from Arabidopsis thaliana. Transgenic petunia plants (cv, Marco Polo Odyssey) harboring cor15a:FAD7 were established and conformed by PCR and Southern analysis. Therefore in our study, FAD7 gene expression was induced by exposure to cold temperatures and down regulated under normal growing conditions. RT-PCR indicated a marked increase in FAD7 expression between transgenic plants exposed to a short (3 days) cold treatment prior to long-term cold storage and those that did not receive a cold induction treatment. Transgenic and wild-type plants were induced in cold (3 °C) for 3 days, returned for normal greenhouse conditions for 5 days and then subjected 3 weeks of continuous cold storage. It was observed that two out of eight transgenic lines showed superior cold tolerance relative to wild-type petunia plants. Additionally, plants that showed cold tolerance completely recovered; growing and flowering normally when returned to the 25 °C greenhouse conditions.
Mariya V. Khodakovskaya, Richard J. McAvoy*, Keming Luo, and Yi Li
The bacterial isopentenyl transferase (ipt) gene involved in cytokinin biosynthesis was fused with a promoter from the serine proteinase inhibiter (win3.12) gene of Populus x generosa and introduced into Nicotiana tabacum (cv. Havana). Transformants were confirmed by PCR reaction and Southern blot analysis, and then analyzed for phenotypic characteristics. RT-PCR analysis detected transcripts of the ipt gene following the wounding of win3.12:ipt transgenic plants. In win3.12: ipt transgenic plants, lateral shoot number and the diameter of lateral branches that developed following apical shoot removal increased relative to wild-type plants. However, the increase in cytokinin production following wounding appeared to be short lived. The potential utility of this construct in enhancing lateral branching in ornamental crops will be discussed.
San-Gwang Hwang, Yi-Ying Li, and Huey-Ling Lin
The king oyster mushroom [Pleurotus eryngii (DC.:Fr.) Quél.] is gaining popularity across the world due to its excellent taste, high nutritional quality, medicinal value, and long shelf life. Conventional substrates for king oyster mushroom cultivation consist of sawdust derived from various tree species. Sawdust demand is increasing worldwide, creating a need for alternative materials that can at least partially replace sawdust as substrate for king oyster mushroom. In Taiwan, as in other countries that grow fruit trees, pruned fruit tree branches are an expensive agricultural waste, particularly if they are not recycled or reused. In the present study, we evaluated substrates containing sawdust and different proportions of material ground from pruned wax apple or Indian jujube branches for cultivation of king oyster mushroom. Our results suggested that among all five substrate mixes tested, the best substitute for conventional sawdust (100% sawdust) was a substrate that contained 75% sawdust mixed with 25% materials ground from trimmed wax apple branches (Wax apple 25%). Furthermore, determination of mineral element content, pH, and electrical conductivity (EC) levels of the substrates both before spawn inoculation and after harvesting revealed no significant changes in mineral content, a slight reduction in pH value, and a minor increase in EC levels after cultivation. Taken together, results from this study suggest that agricultural wastes from pruned fruit tree branches can partially replace sawdust as the cultivation substrate for king oyster mushroom.
Mariya Khodakovskaya, Richard McAvoy, Hao Wu, Jeanne Peters, and Yi Li
Chill injury and leaf senescence occur in plants held in prolonged cold, dark storage. To increase tolerance to these conditions, Nicotiana alata and N. tabacum were transformed with either the FAD7 or IPT genes under the control of a cold-inducible promoter (cor15a). FAD7 encodes for omega-3-fatty acid desaturase and was used to resist cold-stress. IPT encodes the cytokinin-pathway enzyme isopentenyl transferase and was used to delay senescence. Independent FAD7 and IPT lines were crossed to produce double transgenic seed. Seedlings from single transgenic (cor15a-IPT or cor15a-FAD7) lines, double transgenic lines, and the wild-type were exposed to prolonged cold, dark conditions. After 3 months in the dark at 2 °C, survival of independent double transgenic N. tabacum lines ranged up to 80% to 90%. However only 40% of FAD7 seedlings survived, 10% of IPT seedlings survived, and no wild-type plants survived. Double transgenic N. alata seedlings average 90% survival under similar conditions and RT-PCR revealed expression of both the IPT and FAD7 genes. Omega-3-FAD enzyme activity increases desaturation in chloroplast membrane fatty acids. When exposed to prolonged cold, the molecular fraction of polyunsaturated fatty acids (18:3 and 16:3) in leaves of wild-type N. alata decreased while monounsaturated (16:1 and 18:1) and saturated fatty acid species (16:0 and 18:0) increased dramatically. In double transgenic N. alata lines exposed to prolonged cold, the molecular fraction of 18:3 and 16:3 increased, while the 16:0 and 18:0 species decreased dramatically compared to nonchilled double transgenic plants.
Yi Tan, Baisha Li, Yi Wang, Ting Wu, Zhenhai Han, and Xinzhong Zhang
Agrobacterium-mediated genetic transformation is commonly used in dicotyledon plants such as apples. The regeneration ability of the recipient is an important factor in transformation efficiency. Here, the variations in bud regeneration rate (BRR) and the number of adventitious buds (NAB) formed per explant in Malus germplasm accessions with phenological stage were estimated. Both BRR and NAB of explants at the dormancy broken and spring sprouting stages were significantly higher than those at the autumn sprouting stage. The genetic diversity and inheritance of BRR and NAB were evaluated using 153 Malus germplasm accessions and 78 hybrid trees of Jonathan × Golden Delicious. Malus sieversii 31, Liberty, and Smoothee exhibited significantly high BRR (98.33%, 98.33%, and 93.33%, respectively) and a large NAB without vitrification. BRR and NAB linearly correlated with each other but not with callus formation rate. The broad sense heritability of the regeneration rate was 92.16%. The three Malus accessions that had high regeneration ability, and some of their sexual descendants, might be outstanding genetic resources for future genetic transformation.
Li Ma, Chang Wei Hou, Xin Zhong Zhang, Hong Li Li, De Guo Han, Yi Wang, and Zhen Hai Han
Understanding of root growth patterns and architecture of apple (Malus ×domestica Borkh.) trees is very important for commercial apple production. Most commercial apple trees are usually a grafted complex consisting of the scion and the rootstock, each of which is a different genotype. Recently, rootstocks of dwarf tree species have been used extensively to meet the convenience in management; however, this practice appears to negatively impact root development. Using minirhizotrons, we investigated root dynamics, root spatial distribution, and shoot growth in ‘Red Fuji’ scion grown: 1) directly on dwarf and vigorous root stocks and 2) on a dwarf root stock placed in between the non-dwarf scion and non-dwarf rootstock (hereinafter referred to as an interstem). The results showed that: 1) one or two peaks in total root length density (TRLD) were observed in each scion/rootstock combinations every year; 2) the greatest TRLD peaks were always observed in between May and December. The peaks of shoot growth were always asynchronous with that of white root length density; 3) compared with scion/vigorous rootstock combinations, inserting a dwarfing interstem between the scion and vigorous seedling rootstock reduced the TRLD; 4) scion/vigorous rootstock combinations had a relatively deep, widespread and large root system. Scion/dwarfing rootstock combinations had a root system distributed in a small region; and the root systems of scion/dwarfing interstem/vigorous rootstock combinations tended to be intermediate between those of scion/vigorous rootstock and scion/dwarfing rootstock. This implies that the insertion of interstems altered the root architecture by not only the quantity of roots, but also the spatial distribution.
Jiao Chen, De-bao Yuan, Chao-zheng Wang, Yi-xing Li, Fen-fang Li, Ke-qian Hong, and Wang-jin Lu
Many reports indicate that an abundance of really interesting new gene (RING) play key roles in regulating defense responses against abiotic and biotic stresses in plants. In this study, the cloning and functional characterization of a RING gene, MaRING2, in banana (Musa acuminata) fruit are reported. MaRING2 belongs to the NEP1-interacting protein (NIP) RING-H2 finger protein family. Gene expression profiles revealed that MaRING2 was cold responsive and induced by abscisic acid (ABA) treatment during cold storage. In this study, the MaRING2 under control of the Cauliflower mosaic virus 35S (CaMV 35S) promoter was transformed to tobacco (Nicotiana benthamiana) using agrobacterium (Agrobacterium tumefaciens)-mediated transformation. The resultant MaRING2-overexpressing transgenic plants (35S:MaRING2) exhibited significantly increased tolerance to low temperatures and were hypersensitive to exogenous ABA in terms of germination and early seedling growth. In addition, overexpression of MaRING2 enhanced the expression of stress-responsive genes under normal (before cold stress) or cold conditions. These results demonstrate the biological role of MaRING2 in conferring cold tolerance. Taken together, these results suggest that MaRING2, a C3H2C3-type RING protein, is a positive regulator of the ABA-dependent stress response.
Ting Min, Li-Fang Niu, Jun Xie, Yang Yi, Li-mei Wang, You-wei Ai, and Hong-xun Wang
NAC transcription factors have been characterized in numerous plants, and the NAC gene has been shown to be involved not only in plant growth and development, but also in plant responses to abiotic and biological stresses, such as drought, high salinity, low temperature, and anaerobic/hypoxic stress. Creating an environment of anaerobic/hypoxic stress has been shown to be one of the effective storage methods for delaying the browning of fresh-cut lotus (Nelumbo nucifera) root. However, whether NAC is associated with lotus root browning under anaerobic stress has not been studied. In this study, vacuum packaging (VP; anaerobic/hypoxic stress) effectively delayed the browning of fresh-cut lotus root. The changes in the expressions of NnPAL1, NnPPOA, and NnPOD2/3 were consistent with phenylalanine aminolase, polyphenol oxidase (PPO), and peroxidase (POD) enzyme activity changes and lotus root browning. Using RNA sequencing, five NnNAC genes were isolated and studied. Transcriptional analysis indicates that the NnNAC genes showed different responses to VP. The expressions of NnNAC1/4 were inhibited by VP, which was consistent with the observed change in the degree of fresh-cut lotus root browning. However, NnNAC2 messenger RNA (mRNA) levels were upregulated, and the expressions of NnNAC3/5 showed no clear differences under different packaging scenarios. Thus, NnNAC1/4 were identified as promising candidates for further transcriptional regulation analysis in lotus root to understand more fully the molecular mechanism of browning under anaerobic/anoxic stress.
Kai Zhao, Feng Zhang, Yi Yang, Yue Ma, Yuexue Liu, He Li, Hongyan Dai, and Zhihong Zhang
GA20-oxidase (GA20-ox) is a key enzyme involved in the biosynthesis of gibberellic acid (GA). To investigate its role in plant growth and development, we suppressed MdGA20-ox gene expression in apple (Malus domestica cv. Hanfu) plants by RNA interference (RNAi). After 20 weeks of growth in the greenhouse, significant phenotype differences were observed between transgenic lines and the nontransgenic control. Suppression of MdGA20-ox gene expression resulted in lower plant height, shorter internode length, and higher number of nodes compared with the nontransgenic control. The expression of MdGA20-ox in transgenic plants was significantly suppressed, and the active GA content in transgenic lines was lower than that in the nontransgenic control. These results demonstrated that the MdGA20-ox gene plays an important role in vegetative growth, and therefore it is possible to develop dwarfed or compact scion apple cultivars by MdGA20-ox gene silencing.