Materials and Methods

Three pecan cultivars, Pawnee (grafted on ‘Peruque’), Kanza (grafted on ‘Giles’), and Maramec (grafted on ‘Colby’) of 27-year-old trees planted in the same block, were used in this experiment. Samples were collected from an experiment orchard located in Cimarron Valley Research Station (97°02′13″ W, 35°58′55″ N), Perkins, OK, USA. The 1-year-old shoot samples of the three pecan cultivars were collected during the outer bud scale shed stage. Subsequently, the samples were subjected to an 8-hour treatment at –6 °C using a Conviron E8 Freezing Unit in the laboratory. Following the treatment, the shoots were cultivated in a growth chamber, where temperature and humidity conditions were set to replicate spring conditions. Controls were without a freeze temperature treatment; in one set of the controls, the primary buds were removed manually, whereas on a matching set, primary buds were not removed. All shoots were kept in growth chambers for 2 to 3 weeks after the temperature treatments and observed visually.

The top section (4 to 5 cm in length from the terminal bud) from six shoots of each treatment and controls were tested for sugar and starch. The wood and bark of each section were separated, dried and ground first into small pieces using a Willey mill (Arthur K. Thomas Co., Philadelphia, PA, USA), and then to a fine powder in 2-mL tubes with 4-mm bearing balls (Precision Chrome Steel G25; UXCELL, Hong Kong, China) using a Mini-Beadbeater 96 (Biospec Products, Bartlesville, OK, USA). Fine powder samples (25–27 mg) were incubated in 1 mL of ultra-pure water at 70 °C for 15 min followed by centrifugation for 10 min at 15,000 g_n. The supernatant was diluted, and soluble sugars were quantified using anthrone as reagent. For water-insoluble starch analysis, the remaining pallet were washed using 95% ethanol, incubated at 100 °C for 10 min to allow starch gelatinization, and then digested with amylo-glucosidase (700 units/mL), alpha-amylase (70 units/mL), and sodium acetate (0.2 M, pH 5.5) for 4 h at 37 °C in a Roto-Therm^TM Plus Incubated Rotator (H2024; Benchmark Scientific, Sayreville, NJ, USA). Colorimetric anthrone reactions were conducted in 96-well microplates. Absorbance readings were obtained using a microplate reader (Epoch, Bio-TEK, Instruments Inc., Winooski, VT, USA) at 620 nm. Gen5 3.10.lnk software was used to convert absorbance readings into glucose equivalents using a standard curve generated with each 96-well microplate. Each sample was tested three times.

A two-way analysis of variance was performed to test for statistically significant treatment or cultivar effect on carbohydrate content t at the α = 0.05 level using RStudio in R (version 1.4.1106). Tukey’s honestly significant difference (for pairwise comparison) was performed in R (version 1.4.1106).

Results and Discussions

There are two or three compound buds at each node, primary (Fig. 1A), secondary (Fig. 1A), and tertiary buds (sometimes existing). The compound bud contains two catkin buds (male flowers) and a center mixed bud (Smith and Cheary 2010). Pecan is unique in that if the primary bud is killed, the secondary or tertiary buds can develop and bear both pistillate and staminate flowers (Wood and Payne 1983). Figure 1B shows ‘Kanza’ after the primary bud was freeze damaged and the secondary bud continued to develop into a current-year shoot with female flowers. Smith and Cheary (2010) reported pistillate flower production from secondary buds could be 60% less than the primary buds’ production potential. In 2021, after a spring freeze in Oklahoma, many growers recovered 60% to 70% of production in ‘Kanza’ through secondary buds. However, the secondary bud growth in other cultivars, such as Pawnee, Maramec, Lakota, and native pecans was not significant.

(A) Secondary bud remains dormant when primary buds are healthy. (B) Secondary buds in ‘Kanza’ sprout and bear flowers after primary buds were damaged in spring freeze on 21 Apr 2021, at Oklahoma State University experimental orchards in Perkins, OK, USA.

Citation: HortScience 58, 10; 10.21273/HORTSCI17335-23

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(A) Secondary bud remains dormant when primary buds are healthy. (B) Secondary buds in ‘Kanza’ sprout and bear flowers after primary buds were damaged in spring freeze on 21 Apr 2021, at Oklahoma State University experimental orchards in Perkins, OK, USA.

Citation: HortScience 58, 10; 10.21273/HORTSCI17335-23

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(A) Secondary bud remains dormant when primary buds are healthy. (B) Secondary buds in ‘Kanza’ sprout and bear flowers after primary buds were damaged in spring freeze on 21 Apr 2021, at Oklahoma State University experimental orchards in Perkins, OK, USA.

Citation: HortScience 58, 10; 10.21273/HORTSCI17335-23

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In Fig. 2, the sugar content in both the bark (phloem) and wood (xylem) and the starch in the wood of all the treatments and controls in ‘Kanza’ were higher than in ‘Pawnee’ and ‘Maramec’. The sugar content in the ‘Kanza’ wood in all treatments was significantly higher than in ‘Maramec’. Sugar content in ‘Pawnee’ was slightly higher than in ‘Maramec’. These results parallel the secondary budbreak; ‘Kanza’ and ‘Pawnee’ had a higher budbreak than ‘Maramec’ when primary buds were impaired by freeze or primary bud removal (Zhang et al. 2022). Earlier research has demonstrated that in winter and early spring, the xylem functions as the primary carbon source as phloem transport is blocked (Améglio et al. 2002). This supports our observations of the of higher sugar content in the xylem and subsequent higher secondary budbreak, suggesting higher carbohydrate levels support secondary budbreak rate after freeze damage.

The bark soluble sugars, bark starch, wood soluble sugars, and wood starch [all in mg/g dry weight (DW)] from branches treated with –6 °C for 8 h, control with primary buds removed, and control (no treatment) in three pecan cultivars.

Citation: HortScience 58, 10; 10.21273/HORTSCI17335-23

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The bark soluble sugars, bark starch, wood soluble sugars, and wood starch [all in mg/g dry weight (DW)] from branches treated with –6 °C for 8 h, control with primary buds removed, and control (no treatment) in three pecan cultivars.

Citation: HortScience 58, 10; 10.21273/HORTSCI17335-23

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The bark soluble sugars, bark starch, wood soluble sugars, and wood starch [all in mg/g dry weight (DW)] from branches treated with –6 °C for 8 h, control with primary buds removed, and control (no treatment) in three pecan cultivars.

Citation: HortScience 58, 10; 10.21273/HORTSCI17335-23

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The controls of ‘Kanza’ and ‘Pawnee’, which had no freeze treatment, no bud removal, and no secondary budbreak, exhibited higher carbohydrate contents in both wood and bark compared with ‘Maramec’. This pattern of carbohydrate content resembled that of freeze-treated and secondary bud-developed shoots, suggesting that carbohydrate levels regulate and determine secondary budbreak rather than vice versa. In other words, the controls negate the possibility that secondary budbreak leads to high or low carbohydrate levels in shoots because carbohydrates were tested after secondary budbreak in this research.

Pecan has two types of dichogamy: protandry and protogyny. Protandrous cultivars produce anthers and pollen on the male flowers before the stigmas of the female flowers are receptive. Protogynous cultivars producing anthers and pollen on the male flowers after the stigmas of the female flowers are receptive (Kuden et al. 2013). ‘Pawnee’ had a higher budbreak rate than ‘Kanza’ and ‘Maramec’ in the growth chamber. However, in the 2021 freeze, ‘Pawnee’ production did not recover as ‘Kanza’ did through secondary budbreak. Possibly ‘Pawnee’ is protandrous, and its flowering capacity from secondary buds differed from that of the protogynous cultivars Kanza and Maramec. Sparks (1992) and Wells (2008) reported ‘Desirable’, another protandrous cultivar, exhibiting abnormal flowers after the freeze.

Our results suggest that carbohydrate levels in the bud-bearing 1-year-old shoots affect the budburst of pecan’s secondary buds if the primary buds have been damaged by freeze. Therefore, production practices to improve tree carbohydrate levels in the late summer and fall could potentially protect pecan production from spring freeze damage.