Elevated auxin and reduced cytokinin contents in rootstocks improve their performance and grafting success

Abstract

Plant grafting is an important technique for horticultural and silvicultural production. However, many rootstock plants suffer from undesirable lateral bud outgrowth, low grafting success rates or poor rooting. Here, we used a root-predominant gene promoter (SbUGT) to drive the expression of a tryptophan-2-monooxygenase gene (iaaM) from Agrobacterium tumefaciens to increase auxin levels in tobacco. The transgenic plants, when used as a rootstock, displayed inhibited lateral bud outgrowth, enhanced grafting success rate and improved root initiation. However, root elongation and biomass of SbUGT::iaaM transgenic plants were reduced compared to those of wild-type plants. In contrast, when we used this same promoter to drive CKX (a cytokinin degradation gene) expression, the transgenic tobacco plants displayed enhanced root elongation and biomass. We then made crosses between the SbUGT::CKX and SbUGT::iaaM transgenic plants. We observed that overexpression of the CKX gene neutralized the negative effects of auxin overproduction on root elongation. Also, the simultaneous expression of both the iaaM and CKX genes in rootstock did not disrupt normal growth and developmental patterns in wild-type scions. Our results demonstrate that expression of both the iaaM and CKX genes predominantly in roots of rootstock inhibits lateral bud release from rootstock, improves grafting success rates and enhances root initiation and biomass.

Keywords: auxin; cytokinins; grafting success rate; lateral buds release; root growth and development; rootstock.

Figure 1

Root‐predominant expression of an auxin biosynthetic gene (iaaM) inhibited lateral bud release. (a) Histochemical staining of GUS activity in a 5‐day‐old SbUGT::GUS tobacco T₁ seedling, showing that the SbUGT promoter was predominantly active in roots. (b) Four‐month‐old wild‐type and SbUGT::iaaM‐15 tobacco plants, showing that expression of the SbUGT::iaaM gene did not affect growth and developmental patterns of leaves and shoots. (c) Three weeks after decapitation, wild‐type plants released numerous lateral buds, while the SbUGT::iaaM‐39 plants had no lateral buds released from the decapitated shoots; the arrow heads indicate the decapitated shoots of wild‐type (left) and SbUGT::iaaM‐39 (right) tobacco. (d) A 4‐month‐old transgenic tobacco plant overexpressing the iaaM gene under the control of a shoot and leaf tissue active promoter ( SAUR ), displaying strong auxin‐overproducing phenotypes, reduced shoot elongation and leaf epinasty. (e) Expression levels of the iaaM gene in shoot tissues of 2‐month‐old SbUGT::iaaM and SAUR::iaaM tobacco plants. (f) Relatively high expression of the iaaM gene in roots but low expression in shoots was observed in the 2‐month‐old SbUGT::iaaM‐39 plant line. (g) Expression level of the auxin‐responsive GH3 gene was enhanced in roots of SbUGT::iaaM‐39 line. Asterisks (*) represent significant differences between wild‐type and SbUGT::iaaM‐39 tobacco using two‐tailed Student's t‐test with the pooled variance (P < 0.05). Bars represent standard errors.

Figure 2

Using SbUGT::iaaM‐39 plants as rootstock led to inhibited lateral bud release from rootstock stumps and vigorous scion growth. (a) Two weeks after grafting, WT/WT grafts had released lateral buds and scion growth was inhibited. (b) If WT/WT grafts’ lateral buds were manually removed from the rootstock stumps, scions grew vigorously. (c) WT/iaaM grafts had no lateral buds released from the rootstock stumps, and scions grew vigorously. (d) Two months after grafting, WT/iaaM grafts had normal growth similar to WT/WT with lateral buds removed from rootstock stumps. (e) Two weeks following decapitation, scions of WT/iaaM had normal lateral buds release similar to the scions of WT/WT grafts, showing that increase in auxin in rootstock did not affect  normal growth and development of the scions. Circles show scions. Arrow heads indicate released lateral buds. S: scion. L: lateral bud. R: rootstock.

Figure 3

Negative effects of the SbUGT::iaaM gene expression on root growth can be compensated with expression of the SbUGT::CKX gene. (a) Stem cuttings with shoot tips of wild‐type, SbUGT::iaaM‐39 (iaaM) and SbUGT::iaaM‐39/SbUGT::CKX ‐64 (iaaM+ CKX ) hybrid plant after being rooted in fritted clay medium for 10 days, showing that the iaaM gene expression promotes root initiation. (b–d) Six‐week‐old (d) iaaM+ CKX plants had more root growth compared to the (b) wild‐type or (c) iaaM plants. (e) Stem cuttings of SbUGT::CKX‐64 (CKX) plants after being cultured on a MS medium for 3 weeks, showing more and longer roots than wild type. (f) Significant reduced zeatin contents observed in the roots of CKX plants when compared to wild‐type plants. (g) Eight‐day‐old progeny seedlings from the crossings of: iaaM or CKX to wild type, self‐crossed wild type and iaaM to  CKX . The results showed that auxin‐mediated reduction in root growth was neutralized with expression of the CKX gene in roots. (h) Effects of the iaaM and CKX gene expression on primary root length. Asterisks (*) represent significant differences compared to wild type using two‐tailed Student's t‐test with the pooled variance (P < 0.05). Bars represent standard errors.

Figure 4

The SbUGT::iaaM‐39/SbUGT::CKX ‐64 hybrid plant (iaaM+CKX ) used as rootstock inhibited lateral bud release from their stumps and enhanced scion growth. (a–d) Three weeks after grafting, (a) WT/WT grafts had little scion growth because of outgrowth of a lateral shoot; (b) WT/iaaM and (c) WT/iaaM+CKX had no lateral buds released from their rootstock stumps and showed vigorous scion growth; and (d) WT/ CKX had lateral shoot outgrowth with little scion growth. S: scion. L: lateral bud. R: rootstock.