Tetraploid Induction by Colchicine Treatment and Crossing with a Diploid Reveals Less-Seeded Fruit Production in Pointed Gourd (Trichosanthes Dioica Roxb.)
Total Page：16
File Type：pdf, Size：1020Kb
plants Article Tetraploid Induction by Colchicine Treatment and Crossing with a Diploid Reveals Less-Seeded Fruit Production in Pointed Gourd (Trichosanthes dioica Roxb.) Jahidul Hassan 1,*, Ikuo Miyajima 2, Yukio Ozaki 3, Yuki Mizunoe 3, Kaori Sakai 4 and Wasimullah Zaland 1 1 Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka 819-0395, Japan; [email protected] 2 Institute of Tropical Agriculture, Kyushu University, Fukuoka 819-0395, Japan; [email protected] 3 Laboratory of Horticultural Science, Kyushu University, Fukuoka 819-0395, Japan; [email protected] (Y.O.); [email protected] (Y.M.) 4 Laboratory of Agricultural Ecology, Kyushu University, Fukuoka 819-0395, Japan; [email protected] * Correspondence: [email protected]; Tel./Fax: 02-9205333 Received: 20 January 2020; Accepted: 9 March 2020; Published: 17 March 2020 Abstract: Pointed gourd (Trichosanthes dioica Roxb.) (2n = 2x = 22) is a dioecious cucurbit vegetable and green fruit that is edible after cooking. Consumers prefer to consume seedless or less-seeded fruit because seeds are unpalatable due to their hard coats. Therefore, the cross compatibility between the diploid and induced tetraploid will be helpful for seedless or less-seeded fruit production. Thus, the present study was conducted using mature seeds that were immersed in 0.05%, 0.1%, and 0.5% colchicine for 24, 48, and 72 h to induce tetraploids. These tetraploids were used as parents (male or female) in the inter-ploidy and intra-ploidy crosses. A flow cytometric analysis confirmed the induction of three tetraploids at 0.5% colchicine for 48 and 72 h soaking periods. Among these, two (2) females and one (1) male were differentiated after flower initiation. Crossing between the tetraploid’s maternal and diploid paternal parent (4x 2x), which were revealed to be compatible, × resulted in a similar fruit set rate and shape as those of the diploid. In addition, a seed number of 4x 2x produced fruits that were drastically reduced to 1.8 seeds per fruit, whereas the natural diploid × fruits had 26.4 seeds per fruit. These findings suggest that colchicine-induced tetraploid females are important genetic resources for less-seeded fruit production. The genetic stability of tetraploid clones can easily and effectively be maintained by vine cutting for advanced uses. Keywords: colchicine; cucurbits; dioecious; flow cytometry; polyploidization; seed; seedless; tetraploid; Trichosanthes dioica 1. Introduction Pointed gourd (Trichosanthes dioica Roxb.) is one of the most important cucurbit summer vegetables in India and Bangladesh. Genetically, it is a diploid type with a chromosome number of 2n = 2x = 22, where 11 bivalents are observed in the pollen’s mother cells [1]. Botanically, it is a dioecious type, where the male and female flowers are carried on separate plants. Due to its dioecism, cross pollination is inevitable for fruit setting. Immature green fruits containing seeds with soft coats are edible after cooking. Usually, each fruit contains more than 20 seeds and becomes harder at 3–4 weeks after pollination (Figure1). At this point, it loses consumer acceptance because of its unpalatable seeds with hard coats. Parthenocarpy induction approaches might be able to solve this problem. Plants 2020, 9, 370; doi:10.3390/plants9030370 www.mdpi.com/journal/plants Plants 2020, 9, x FOR PEER REVIEW 2 of 19 are edible after cooking. Usually, each fruit contains more than 20 seeds and becomes harder at 3–4 weeks after pollination (Figure 1). At this point, it loses consumer acceptance because of its unpalatable seeds with hard coats. Parthenocarpy induction approaches might be able to solve this problem.Plants 2020, 9, 370 2 of 16 Figure 1. FruitsFruits at at the the edible edible stage stage with with soft-coated soft-coated seeds seeds ( (AA)) and and hard-coated hard-coated seeds seeds ( (BB).). The The bar bar indicatesindicates 1 1 cm. cm. The inductioninduction ofof parthenocarpy parthenocarpy by exogenousby exogenous plant plant growth growth regulators regulators has already has beenalready reported been reportedfor pointed for gourdpointed [2 ]gourd and other [2] and cucurbits other cucurbits [3–5]. However, [3–5]. However, this practice this ispractice not genetically is not genetically stable or stableeconomically or economically feasible because feasible itbecause involves it involves extra investment extra investment in laborand in labor chemicals and chemicals every year. every Another year. Anotherapproach approach to manage to this manage problem this is problem to develop is triploidto develop clones triploid after crossing clones after between crossing tetraploid between and tetraploiddiploid parents. and diploid From aparents. horticultural From pointa horticultura of view,l such point triploids of view, are such expected triploids to beare superior expected to to their be superiordiploid counterparts,to their diploid with counterparts, a higher yield, with morea high vigorouser yield, growth,more vigorous and seedlessness growth, and or seedlessness less-seeded orcharacteristics, less-seeded characteristics, making them moremaking attractive them more to consumers attractive [6 to]. Itconsumers has been reported[6]. It has that been a tetraploid reported thatpumpkin a tetraploid fruit’s pumpkin weight is fruit’s 2.9 kg weight with fewer is 2.9seeds kg with (30), fewer while seeds that (30), of diploids while that is 2.2of diploids kg with moreis 2.2 kgseeds with (122) more [7]. seeds However, (122) [7]. this However, technique this is restricted technique for is seed restricted propagated for seed species propagated due to thespecies inherent due todi ffitheculties inherent in maintaining difficulties in genetic maintaining stability, genetic as well stability, as their as low well rate as oftheir viable low seed rate productionof viable seed [8]. productionHowever, this [8]. seedlessness However, strategythis seedlessness does not a ffstrategyect the commercial does not utilityaffect andthe multiplicationcommercial utility of pointed and multiplicationgourd because of of pointed vegetative gourd propagation because of by vegetati vine cutting.ve propagation It is hypothesized by vine cutting. that colchicine It is hypothesized treatment thatmight colchicine be effective treatment in inducing might tetraploidsbe effective inin pointedinducing gourd. tetraploids These in inducedpointed tetraploidsgourd. These (female induced or tetraploidsmale) are assumed (female or to male) be cross are compatible assumed to with be cross diploids compatible (male or with female), diploids and (male their fruitsor female), would and be theirless-seeded fruits would or seedless be less-seeded (Figure2). In or addition, seedless if (Figur these fruitse 2). In produce addition, viable if these seeds, fruits then theseproduce seeds viable will seeds,be F1 and then could these be seeds used will to determinebe F1 and could the ploidy be used level. to determine If these progenies the ploidy are level. recognized If these as progenies triploids, arethen recognized it would be as confirmed triploids, then that colchicine-inducedit would be confirmed polyploidization that colchicine-induced in pointed gourdpolyploidization is genetically in pointedinheritable gourd and is that genetically these triploids inheritable would and be sterile.that these When triploids the sterile would female be flowerssterile. When cross withthe sterile fertile femalediploid flowers male flowers, cross thewith pointed fertile gourd diploid produced male flowers, would be the completely pointed seedless.gourd produced Once triploid would line be of completelythe pointed seedless. gourd is developed,Once triploid it canline thenof the be poin multipliedted gourd by vineis developed, cutting for it commercialcan then be productionmultiplied byand vine distributed cutting for to localcommercial nurseries. production Further, ifand F1 seedsdistributed are not to viable,local nurseries. then tetraploid Further, lines if F will1 seeds also are be notvery viable, important then fortetraploid producing lines less-seeded will also be fruits very while important being for cross-compatible producing less-seeded with diploids. fruits Thesewhile beingfacts will cross-compatible help facilitate thewith development diploids. These of the facts tetraploid will help line facilitate as the the logical development first step for of the tetraploid expansion lineof genetic as the resources logical first in further step for triploid the expansion breeding programs of genetic for resources pointed gourd. in further triploid breeding programs for pointed gourd. Colchicine is considered an effective chromosome doubling agent for inducing polyploidy in many vegetables [9]. Hazra [10] observed colchiploid induction in pointed gourd when 0.2% Plants 2020, 9, x FOR PEER REVIEW 3 of 19 colchicine was applied to five-day-old seedlings, but this resulted in an abnormal morphology and an inability to produce flowers or fruit. However, the effectiveness of colchicine depends highly on Plantsthe2020 concentration, 9, 370 applied, the duration of treatment, the type of explant, and the penetration3 of 16of the compound [11]. Figure 2. Hypothetical pathways of polyploidization and triploid breeding for seedless fruit production Figure 2. Hypothetical pathways of polyploidization and triploid breeding for seedless fruit in pointed gourd (Trichosanthes dioica Roxb.). (A) Mature seeds