Chrysanthemum: Advances in Tissue Culture, Cryopreservation, Postharvest Technology, Genetics and Transgenic Biotechnology

Biotechnology Advances 21 (2003) 715–766 www.elsevier.com/locate/biotechadv

Research review paper Chrysanthemum: advances in tissue culture, cryopreservation, postharvest technology, genetics and transgenic biotechnology

Jaime A. Teixeira da Silva *

Faculty of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa 761-0795, Japan Received 26 March 2003; accepted 22 July 2003

Abstract

Members of the Chrysanthemum-complex include important floricultural (cut-flower) and ornamental (pot and garden) crops, as well as plants of culinary, medicinal and (ethno)- pharmacological interest. The last 35 years have seen a tremendous emphasis on their in vitro tissue culture and micropropagation, while the latter 10–15 years has seen a surge in transformation experiments, all aimed at ameliorating aesthetic and growth characteristics of the plants. This review highlights all available literature that exists on ornamental Chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation. D 2003 Elsevier Inc. All rights reserved.

Keywords: Chrysanthemum; Cryopreservation; Genetic transformation; Postharvest technology; Tissue culture

1. Introduction

Chrysanthemum is one of the most important global cut flower and pot plants. Commercial cultivars are usually cultivated by vegetative cuttings or suckers. Traditional breeding, and more recently, together with genetic, molecular techniques, has focused on the enhancement of the plant’s ornamental value through the improvement of flower colour, size and form, vegetative height, growth form and sensitivity to light quality/

Abbreviations: AA, aminoglycoside antibiotic; PA, polyamine; PGR, plant growth regulator; SAAT, sonication-assisted Agrobacterium transformation; TCL, thin cell layer; TSL, threshold survival level; TSWV, tomato spotted wilt virus. * Tel./fax: +81-72-726-8178. E-mail address: [email protected] (J.A. Teixeira da Silva).

Table 1 Chrysanthemum [Chrysanthemum or Dendranthema(um)] regeneration studies Principal cultivar(s) + Explant sourceE TCL GD Organ No. O/E* Medium PGR Reference others composition*1 ..Tier aSla/BoehooyAvne 1(03 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. (MS basal) Chrysanthemum Bronze Pride Stem No – C, R, Various 2 NAA 0.8 K Hill, 1968 shoot n.s. Kiyomizu Shoot tip No – C, R, n.s. Heller’s/LS/ Tsukamoto and shoot Harada + 1 Fujime, 1971 K 2 IAA 0.1 GA C. morifolium Indianapolis Shoot tip No – Shoot n.s. 0.8K 0.5 IAA + Ben-Jaacov and White Giant #4 10% CM Langhans, 1972 C. cinerariaefolium #4331 Capitulum No – Shoot n.s. 0.001 BA Roest and Bokelmann, 1973 C. makinoi n.s. Embryo rescue No – Plant n.s. n.s. Tanaka and Watanabe, 1972 C. morifolium Bonnie Jean, Tubular floretE Yes Yes C, R, 4–92% 2.1 BA 0.02 Iizuka et al., 1973 Yellow Tuneful + 1 shoot IBA 0.02 IAA C. morifolium Indianapolis White Shoot tip No – Shoot 5 LSz +2 K Earle and Langhans, Giant #4 0.02 NAA 1974a,b C. cinerariaefolium n.s. Shoot, root No – Callus, n.s. 0.05 2,4-D Chumsri and Staba, root 1975 C. morifolium Super Yellow, Bravo Flower pedicel No Yes Shoot 3–4 Liquid MS + Roest and 0.01 IAA Bokelmann, 1975 0.01 BA C. morifolium Shining Light Stem No – Callus n.s. 1 K 1 NAA Bannier and 10% CM Steponkus, 1976 C. morifolium Bravo Leaf, pedicel No – Shoot 1.5–30.5 PGRs n.s.; Broertjies et al., X-ray induced 1976 C. morifolium Indianapolis Shoot tip, petalE No/ – C, shoot n.s. 10 K 1 NAA Bush et al., 1976 yes or PGR-free C. morifolium Kayo¯-no-sakura, Stem No Yes Shoot 67–95% 1–3 IAA 1–3 K Miyazaki et al., Azumajishi + 1 1976 C. boreale/makinoi C. japonense (x4), Ovary culture No Yes Callus n.s. 2 IAA 2 K Watanabe, 1977 C. ornatum +1 C. cinerariaefolium n.s. Shoot tip No – Shoot n.s. 1 NAA 1 BA Grewal and Sharma, 1978 C. coronarium n.s. Seed No – Callus n.s. 1 2,4-D Kohno et al., 1978 C. morifolium Kayo¯-no-sakura, Stem, shoot tip No – Shoot 67–95% 1–3 IAA 1–3 K Miyazaki and Tashiro,

Azumajishi + 1 1978, 1979 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. C. morifolium Blue bird, Montana + 2 Shoot tip, flowerE No/yes – Shoot 100% – Wang and Ma, 1978 C. morifolium Shin Dong Shoot tip No – Shoot n.s. – Lee et al., 1979 C. hortorum Winter/Yellow/Dark Meristem No – C, shoot 2.1–2.9 1 BAP/K 1 IAA Ahmed and Be´la, Westland + 4 1980 C. morifolium Super Yellow, Meristem, No Yes Shoot, root n.s. – Lazar and Cachita, Blanche Poitevine peduncle 1982, 1983; Lazar et al., 1981 C. morifolium White Spider Mesophyll No – Callus 62% 0.001 BAP Schum and Preil, 1981 protoplast C. morifolium Indianapolis White Leaf No – C, shoot 33.5% LSz + 0.8 IAA Sutter and Langhans, Giant #4 0.5 K ! 2K 1981 0.02 NAA C. cinerariaefolium Clone 4331 Shoot tip No – Shoot 2.8–21.5 0.02–2 NAA Wambugu and 0.2/2 BA 0.02 Rangan, 1981 IAA 1–5 K C. morifolium White Spider Axillary bud No – Shoot n.s. 0.1–0.5 K Dabin and Choiseg, 0.05–1 GA3 1983 C. morifolium Birbal Sahni Leaf, stem, root No – Shoot n.s. – Prasad et al., 1993 C. cinerariaefolium + C. coccineum Leaf,stem,floretE No/yes – Callus n.s. 0.5 2,4-D 0.5 BA Zieg et al., 1983 Golden Mass C. cinerariaefolium n.s. Shoot tip No – Shoot n.s. 0.1 2,4-D 3 BA Zito et al., 1983 C. morifolium 4x Marble, 2x Spider, Stem node No – Shoot 2–3.4 0.05–0.5 BA de Donato and Snowdon 0.01–0.5 NAA Peruceo, 1984 C. indicumx1 C. zawadskii x1 Protoplast No Yes Callus 0–53% Various basal Okamura et al., media (9); 1984 varying ammonium (continued on next page) 717 718

Table 1 (continued) Principal cultivar(s) + Explant sourceE TCL GD Organ No. O/E* Medium PGR Reference others composition*1 (MS basal) ..Tier aSla/BoehooyAvne 1(03 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. C. cinerariaefolium Ecuadorian cv. n.s. n.s. No – Shoot 1.1–3.5 20 BA + Staba et al., 1984 varying effects of light/darkness Chrysanthemum sp. n.s. Leaf No – Shoot n.s. – Chen et al., 1985 C. hortorum Pink Camino, Shoot tip/axillary No – Shoot n.s. – Gertsson and Super Yellow + 1 bud Andersson, 1985 C. cinerariaefolium Pyrethrin lines Leaf, petiole No – Callus n.s. 0.5 2,4-D 0.75 K; Kueh et al., 1985 9.4 K / 0.3 BAP 0.01 IAA C. morifolium Shuhou-no-chikara Stem protoplast No – Shoot < 10.5% 2 NAA 1 BA Otsuka et al., 1985 D. grandiflora Snowdon, Altis, Meristem/shoot No – Shoot 60–100% 0.1 BAP 0.05 Ahmed, 1986; Blanche + 3 tip NAA/0.05 BAP Ahmed and 0.025 IAA; heat Andrea, 1987 C. morifolium White Spider Pedicel, petalE No/ – Callus n.s. 1 BA 0.1 IAA de Jong and yes Custers, 1986 D. grandiflorum Shuhou-no-chikara Leaf, stem No – C, R, < 4 0.5/1 NAA Fukai and Oe¨, 1986; shoot 2/5 BA Fukai et al., 1987 Chrysanthemum sp. Shoot tip No – Root < 92% 5 BA 0.02–2 Sun and Li, 1987 NAA/2 IBA C. morifolium n.s. Shoot tip, leaf No – Shoot n.s. – Widiastoety, 1987 Chrysanthemum sp. Shuhou-no- PetalE Yes – Shoot n.s. 10 IAA 10 Ohishi and Sakurai, chikara + 20 cvs. BAP 0.1 K 1988 C. cinerariaefolium HSL 801 Leaf No Yes C, n.s. PGR-free / Paul et al., 1988 (lines C5, C9, C10) shoot 1IBA1NAA; pyrethrum leaf>stem C. morifolium Birbal Sahni Leaf, stem, root No - Shoot 2.4–4.8 0.5–2 K/BAP Prasad and 0.1–0.5 IAA/NAA Chaturvedi, 1988 C. morifolium Blanche Poitevine Meristem tip No Yes Shoot n.s. 0.02 NAA 2 K Votruba and Supre`me + 5 Kody´tek, 1988 C. morifoliumx3 C. coronarium x1 Protoplast No Yes Callus < 39% 0.2/1 BA 0.2/1 NAA Amagasa and Kameya, 1989 C. morifolium Early Charm Leaf, petalE No/ – C, shoot 6–8 10 K 1 NAA Khalid et al., 1989 yes C. morifolium Elegance, Ryember, Protoplast, Yes Yes C, shoot < 50 0.5–4 NAA Malaure et al., Klondike + 10 ray floretE 0.5–4 BAP ! 0.03 1989, 1991a,b

K 0.01 IAA 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. C. cinerariaefolium n.s. (high pyrethrin lines) Leaf No – Callus 100% 5 K 2 2,4-D Ravishankar et al., 1989 D. grandiflora Bluechip + 4 + hybrids Embryo (rescue) No Yes Plantlet 0–54% PGR-free Anderson et al., 1990 C. morifolium Birbal Sahni Leaf, stem No – Shoot 2.15–4.5 2 2,4-D ! Bhattacharya et al., 0.1 IAA 0.2 BAP 1990, 1994 D. grandiflora 1610, Parliament + 8 Leaf No Yes Shoot 5.3–16.8 0.03 IAA 1 BA de Jong et al., 1990 C. morifolium Yuzawa, Enmeiraku, Leaf, stem, flowerE No/ Yes Shoot 2–60% 3 IAA 1 K + 25 Endo et al., 1990, YS + 1 yes para-fluorophenylalanine 1991 (PFP) C. coccineum n.s. AcheneE, petalE Yes – Shoot n.s. 5 BAP 5 NAA Fujii and Shimizu, 1990 D. grandiflorum Shuhou-no-chikara Shoot tip No – Shoot 18% 0.1 BAP 1 NAA + Fukai, 1990; Fukai 5% DMSO; and Oe¨, 1990 cryoprotected D. grandiflora Royal Purple + 10 Leaf, stem No Yes Shoot 0–90% 1–10 BAP Kaul et al., 1990 0.1–5 NAA D. grandiflora Moneymaker Pedicel No – Shoot n.s. 1 K 0.1 IAA ! 1 Lemieux et al., 1990 K 10% CW ! 1K C. morifolium Royal Purple Stem No – Shoot 14.6 0.5–2 BAP 0.2–2 NAA Lu et al., 1990 D. grandiflora n.s. Leaf, stem No – C, shoot n.s. – Rademaker and de Jong, 1990 C. morifolium Pennine Red Shoot tip No – Shoot 100% 0.1 BAP 0.01 NAA Roberts and Smith, 1990 C. hortum 29 clones Mesophyll No Yes Shoot, SE 2.5% 1 NAA 5 K (C); 0.5 Sauvadet et al., protoplast NAA 2 BAP (S) 1990 C. coronarium Mikokui Leaf protoplast No – Callus 7–23% 1/2 MS + 1 NAA Tanimoto and 0.1 BA/Z Kagi, 1990 719 (continued on next page) 720

Table 1 (continued) Principal cultivar(s) + Explant sourceE TCL GD Organ No. O/E* Medium PGR Reference others composition*1 (MS basal) ..Tier aSla/BoehooyAvne 1(03 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. C. cinerariaefolium n.s. 3-year-old plants Axillary bud No – Shoot n.s. 1/2 RT + 1 2,4-D Zito and Tio, 1990 D. indicum Korean Leaf, stem, pedicel No – Shoot 1–2 0.2 IAA 3–10 Ledger et al., 1991 BA 0.1–3 TDZ D. morifolium Peach Margaret + 4 Leaf, stem, pedicel No Yes Shoot < 1 0.2 IAA 3–10 Ibid. BA 0.1–3 TDZ D. grandiflora Iridon, Fortune, Leaf No Yes SE 0–3.7% 1 2,4-D 0.2 BA May and Trigiano, Goldmine + 2 1991 D. grandiflorum Shuhou-no-chikara Leaf No – C, S, SE < 35% 1 NAA 0.5 BA or Shinoyama et al., 1–4 2,4-D 0.1–2 K 1991, 1997, 2002 D. grandiflora Parliament + 13 Leaf No Yes Callus All sizes 0.03 IAA van Wordragen et al., 1991 C. morifolium Fredyule Leaf, stem, flowerE No/ –C,R, n.s. 2/4 IAA 0.5/2 K Bajaj et al., 1992 yes shoot 0.2 NAA B5 + 2 2,4-D C. hortorum n.s. Stem No – Shoot n.s. 2.5 K 0.5 NAA Corneanu and Corneanu, 1992 C. morifolium n.s. ReceptacleE Yes – Shoot n.s. 1 BAP 0.1 NAA Hattori, 1992a,b D. grandiflora Parliament Leaf No – R, shoot n.s. 0.003 IAA van Wordragen et al., 1992a D. grandiflora 1610, Parliament + 6 Leaf No Yes Callus All sizes 0.003 IAA van Wordragen et al., 1992b D. grandiflora Moneymaker Leaf No – Shoot 2–4.8 2 BA 1 NAA Courtney-Gutterson et al., 1993 D. grandiflora 1610, Parliament + 6 Leaf No Yes Shoot 2–4.8 0.003 IAA or 4 de Jong et al., 1993 NAA 1 BA C. cinerariaefolium HSL 801, SL 821 Shoot tip No – Callus 100% 0.5 2,4-D 0.5 BA Dhar and Pal, 1993 C. cinerariaefolium n.s. Callus No – Callus n.s. 0.5 2,4-D 0.5 BAP Krishna et al., 1993 D. grandiflora y Protoplast No – Plants 45–85% 1 NAA 0.5 Z ! 3 Lindsay and Ledger, BAP 0.2 IAA 1993 D. grandiflora Super White Stem, in planta inject No – Shoot n.s. 2 BAP 1 IAA Lowe et al., 1993 D. morifolium Otome zakura, Shoot tip No Yes Shoot < 31 1.5 BAP 0.5 IAA Prasad et al., 1993 Pandhari + 2 D. grandiflora Carillon + 5 Leaf, stem, pedicel No Yes Shoot 0–70% 2 BAP 1 NAA Renou et al., 1993 Chrysanthemum sp. n.s. n.s. No – Root 88% 0.01–1 Tian et al., 1993 paclobutrazol

D. grandiflora Moneymaker Leaf No – Shoot n.s. 2 BA 1 IAA Courtney-Gutterson 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. et al., 1994 D. grandiflora Hatsuyuki, Nogioh + 13 FloretE, petalE Yes Yes Shoot < 64% 1 IAA 1 BAP 5 K Mizutani and Tanaka, 1994 D. grandiflora White Snowdon + 3 Leaf No Yes SE 0.3–0.5 P3–43 + 3 2,4-D Pavingerova´ et al., 1K 1 BAP 1994 5 2iP 10 GA3 D. grandiflora 8382, 89100, 89124 Pedicel No Yes Shoot n.s. 1 BA 1 IAA de Jong et al., 1994 D. grandiflorum Apricot Marble Shoot tip No – Shoot 41–86% 0.1/1 BA 1 IAA + Fukai et al., 1994 cryopreserved D. grandiflora Hekla, Iridon, Polaris Leaf No Yes Shoot n.s. 2 IAA 0.1–0.5 Urban et al., 1994 BA 0.1 NAA D. grandiflora White Snowdon Leaf No – SE 0.3–0.5 P3–43 + 3 2,4-D Benetka and 1K 1 BAP Pavingerova´, 1995 5 2iP 10 GA3 D. grandiflora 1581 Pedicel No – Shoot n.s. 1 BA 0.1 IAA de Jong et al., 1995 (CHR 04) D. grandiflora Orange Regan Stem No Yes Shoot n.s. 0.3–12 BA de Oliveira et al., 0.3–11 K 1995, 1996 0.014–1.4 NAA C. morifolium Bornholm, White Leaf No Yes Shoot n.s. 1/2 MS + 2 BA 0.5 Dolgov et al., 1995 Hurricane NAA ! 0.3 IBA D. grandiflorum 1581 + 9 Stem No Yes Shoot 18.5 1 BA 0.1 IAA Fukai et al., 1995 D. grandiflora Early Charm, Leaf No Yes Shoot 26–88% 1 BA 0.5 NAA Khehra et al., 1995 Tone Maid D. maseimum n.s. FlowerE Yes – Shoot n.s. – Kumar and Kumar, 1995 (continued on next page) 721 722

Table 1 (continued) Principal cultivar(s) + Explant sourceE TCL GD Organ No. O/E* Medium PGR Reference others composition*1 (MS basal) ..Tier aSla/BoehooyAvne 1(03 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. D. grandiflora Iridon, Polaris + 4 Leaf, stem No Yes Shoot 2–10 0.1 NAA 1 BA Yepes et al., 1995, 1999 C. cinerariaefolium HY C,D SY A,B Leaf, stem, flowerE No/ – Callus 4 NAA 0.4 BAP ! Barthomeuf yes 1/2 MS + 4 et al., 1996 NAA 0.4 BAP C. coronarium n.s. Root, hypocotyl, leaf No n.s. Embryo 3–38% 0.1 BA 1 NAA Oka et al., 1996, 1999 C. grandiflora Parliament + 4 Leaf No Yes Shoot n.s. 1 BAP 0.1 NAA 0.5 GA Dolgov et al., 1997 D. grandiflorum YS, Liliput Shoot tip No Yes Shoot 11–100% 0.05–0.2% Colchicine + Endo and Inada, 1 IAA 3 K 1997 C. coronarium n.s. Leaf No – Shoot 73% 0.6 BA 0.5 NAA 6 Lee et al., 1997 AgNO3 ! 1–5 IBA C. morifolium n.s. Shoot tip No – Shoot 95% 0.1 IBA (following Rout and Das, encapsulation) 1997 C. morifolium Deep Pink Leaf, stem No – Shoot 81–92% B5 + 2.5 BAP 1 IAA ! Rout et al., 1997 0.25 IBA/IAA D. grandiflorum Peach Margaret Leaf No – Shoot n.s. 1.9 NAA 1.1 BAP Boase et al., 1998a D. grandiflorum Peach Margaret + 2 + 1y Leaf No Yes Callus n.s. 1.9 NAA 1.1 BAP or Boase et al., 1998b 0.2 IAA 3 BAP C. morifolium n.s. Stem No – Shoot n.s. No PGRs: various de Argollo et al., nitrogen sources 1998 C. cinerariaefolium n.s. Axillary bud No – Shoot n.s. No PGR Chen et al., 1998 D. grandiflora 001 Leaf, stem No – Shoot n.s. 0.5 BA 0.1 NAA Fu et al., 1998 D. grandiflora Fashion Yellow, Leaf No Yes Shoot 100% 2 NAA 0.5 BA Kim et al., 1998a Golden Glory Dendranthema Hybridy Stem No – Shoot 100% 0.2 IAA 3 BA Kim et al., 1998b D. grandiflora Hekla, Iridon, Polaris Leaf No No Shoot 0.1–2.6% Mum B + 0.5 2,4-D ! 2 Sherman et al., K 0.02 NAA 10 GA3 1998b D. grandiflora Shuhou-no-chikara + 22 Stem No Yes Shoot 0–100% 2 NAA 0.2 BA or Takatsu et al., 1998 2 IAA 0.2 BA C. morifolium Aboukyu PetalE Yes – Embryo 0–56% SE 1/10 IAA 0.01–10 Tanaka et al., 1998 Callus 9–90% C BAP 0.1 K Root 0–46% R Shoot 0–80% S

D. grandiflora Fashion Yellow, Leaf No Yes Shoot 30–38% 2 NAA 0.5 BA Young et al., 1998 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. Golden Glory C. morifolium Colchi Bahar + 16 FloretE Yes Yes Shoot 0–5.2 0.2–2 NAA Chakrabarty et al., 0.5–5 BAP 1999 C. cinerariaefolium High pyrethrum lines Flower headE Yes – Callus 100% 1 NAA 1 BAP George et al., 1999 (static culture) C. cinerariaefolium High pyrethrum lines Flower headE Yes – Callus 60–95% 0.2–4 NAA Hitmi et al., 1999b 0.2–41 BAP D. grandiflora n.s. Leaf No – Shoot 100% 0.5 BA 0.1–2 NAA Lee et al., 1999 C. morifolium n.s. Stem No – Shoot n.s. PGR-free Shao et al., 1999 D. grandiflorum Yamabiko Stem No – Shoot n.s. 1/2 MS + 0.2 IAA Takatsu et al., 1999 or 2 NAA 0.2 BA D. grandiflora Lineker, Leaf, stem No Yes Shoot 0–8 0.1 IAA 1 NAA Annadana Moneymaker + 35 0.1–2 BAP 3/10 et al., 2000 GA3 10% CW C. morifolium Colchi Bahar Ray floretE Yes – Shoot 100% 0.2 NAA 1 BAP Chakrabarty et al., 2000 C. morifolium Lilith Ray floretE Yes – Shoot 100% 0.2 NAA 1 BAP Dwivedi et al., 2000 C. morifolium Purnima, Ray floretE, No/ No Shoot < 60 0.2 NAA 0.5 BAP; Mandal et al., Colchi Bahar, Maghi shoot tip yes Gamma ray induced 2000a,b C. morifolium Shuhou-no-chikara Stem No – Shoot 20–85% 0.2 BA (cryopreserved) Sakai et al., 2000 D. grandiflorum Kanseisetsu Stem No – Shoot 16–17 0.1–0.5 NAA 0.5–2 BA Shirasawa et al., 2000 D. grandiflorum Aboukyu Leaf, ray floretE No/ – Embryo 0–58% SE 1/10 IAA 0.01–10 Tanaka et al., yes Callus 0–100% C BAP 0.1 K 2000 Root 23–83% R Shoot 0–58% S 723 (continued on next page) 724

Table 1 (continued)

Principal cultivar(s) + Explant sourceE TCL GD Organ No. O/E* Medium PGR Reference 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. others composition*1 (MS basal) Dendranthema Hybridy Stem No – Shoot n.s. 1.25 NAA 1.25 BA Tosca et al., 2000 D. grandiflora 1581 Stem No – Shoot n.s. CHR 04 Annadana (de Jong et al., 1995) et al., 2001 Chrysanthemum sp 28 different genera/ Disk, ray floretE Yes – Shoot V11 0.2 NAA 2 BA ! Datta et al., 2001a,b species/cvs. 1/2 MS 0.1 NAA D. grandiflora Iridon Leaf No – Shoot Reduction 0.75 BA 2 IAA + George and Tripepi, < 4 ml/L PPMk 2001 D. grandiflora Iridon Leaf, internode No – Shoot n.s. 0.5 BA 1 NAA Zheng et al., 2001 D. grandiflorum Cheonsu Leaf No – Shoot n.s. 0.25–2 BA 2 NAA/IAA Jeong et al., 2002 D. grandiflorum Shuhou-no-chikara + 3 Leaf No – Shoot n.s. 1 BAP 2 NAA Kudo et al., 2002 D. grandiflorum Shuhou-no-chikara, Stem Yes No Embryo 0.01% Embryo 2–4 IAA Teixeira da Silva, Lineker or 4 2,4-D 2003a,c Callus 100% Callus 1–4 TDZ Root 100% Root 1–2 IBA or 2 NAA or 10–20% CW Shoot 2.4–3.2 Shoot 1–2 BA y = D. zawadskii Â D. Â grandiflora (Autumn-flowering chrysanthemum). TCL = thin cell layer; DMSO = dimethyl sulphoxide; GD = genotype dependence; O/E = organ per explant; *= percentages represent the number of explants forming organs; *1 = PGR values in mg lÀ 1 ; K = kinetin; BA= 6-benzyladenine; BAP=(N3)6- benzylaminopurine; 2iP = 6-(dimethylallylamino)-purine; 2,4-D = 2,4-dichlorophenoxyacetic acid; IBA= Indole-3-butyric acid; IAA= 3-indole acetic acid; NAA= a- naphthalene acetic acid; Z = Zeatin; CW/CM = coconut water/milk; PPM = Plant Preservative Mixturek; C = callus, R = roots, S = shoots, SE = somatic embryos; z = Linsmaier and Skoog basal medium. n.s. = not specified. J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 725 quantity (Rout and Das, 1997). Classical breeding has limitations: a restricted gene pool, incompatibility due to parental ploidy differences and the polygenic nature of growth and flowering. Transgenic biotechnology of chrysanthemum overcomes these limitations and allows for more extensive and diverse crop improvement by allowing the introduction of traits coded for by genes across taxonomic barriers.

1.1. History, cultural and medicinal practices

Chrysanthemum, Dendranthema Â grandiflora (Ramat.) Kitamura (Compositae), also known as florist’s chrysanthemum or Higo-giku (in Japanese), originally came from the Greek krus anthemon, meaning gold flower, originating in China (where it has been cultivated for over 2000 years) and Japan from C. indicum var. procumbens Â Chrysan- Chrysanthemum zawadskii var. latilobum (Fukai et al., 1991; Fukai, 1995).Higo chrysanthemum is in nature a bedding flower once exclusively cultivated in Higo (formerly Kumamoto Prefecture) of Kyushu Island, is characterized by unique cultivation and arrangement techniques established in the latter part of the 17th century, based on Confucianist principles where three rows of cultivars represent Heaven (back row), Man (central row) and Earth (front row) (Murayama, 1974). C. coronarium and C. segetum are widely distributed in the Mediterranean, western Africa and Asia (Trehane, 1995). C. coronarium var. coronarium is an ornamental, often found as a common weed, while C. coronarium var. spatiosum is used as a Chinese vegetable (chop-suey). Green leaves and stems of C. segetum are also consumed as vegetables. This culturally rich flower is also globally the second economically most important floricultural crop following rose, and one of the most important ornamental species. The production value of flowers in Japan has more than doubled in the last decade as a result of the rapid improvement of living conditions and a greater enjoyment of life, with chrysanthemums occupying 35% of the total cut-flower production. In terms of chrysanthemum stem production per year (Boase et al., 1997), Japan is the leading producing country (2 billion), followed by the Netherlands (800 mil.), Colombia (600 million), Italy (500 million) and the USA (300 million), while chrysanthemums are the second most important cut flower by sales value (UK market; Flowers and Plants Association, 2001). The use of cut flowers is extensive in Japan: 40% in gift-use, 25% for commercial facilities (hotels, events), 25% in home-use, including religious decorations for Buddhist practices, and 10% for educational purposes in teaching flower arrangement (ikebana). Even though traditional flower arrangement is extensive in Europe, Korea and Japan, their styles differ as follows: in Korea, line and space is emphasized with harmony in nature symbolized by three flower stems; in Japan, line and branch angles of the three cut stems are emphasized through artificial skill; in Europe, colour and mass are emphasized as a geometric composition of the artistic principle (Kim et al., 1991). Chrysanthemums are horticulturally classified into six plant forms: sprays, specimens, cushions, charms, cascades and lilliputs (Boase et al., 1997). Garland chrysanthemum, C. coronarium, cultivated in Japan, China and Southeast Asia, is a closely related to lettuce, and is a valuable edible species (Oka et al., 1999). Chrysanthemum as well as other members of the Anthemideae, is a source of various valuable secondary metabolites, biologically active compounds and essential oils (Schwinn et al., 1994). 726 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766

1.2. Taxonomy and nomenclature

Some division surrounds the exact nomenclature of chrysanthemum (Chrysanthemum sensu stricto being C. carinatum, C. coronarium and C. segetum). Although some efforts have been made to try and classify chrysanthemums, it is a group that still includes Achillea, Ajania, Anthemis, Arctanthemum, Argyranthemum, Artemisia, Balsamita, Chrysanthemum, Dendranthema, Heteranthemis, Hymenostemma, Ismelia, Leucanthemella, Leucanthe- mum, Matricaria, Nipponanthemum, Pyrethrum, Tagetes, Tanacetum and Tripleuspermum, sometimes collectively termed the Chrysanthemum-complex (Asteraceae–Anthemideae; Harborne et al., 1970; Hutchinson et al., 1992; Khallouki et al., 2000). Despite the diversity of use of nomenclature (Table 1), which will certainly not be resolved in the pages of this review, names have been used in this review exactly as those used by referenced authors, irrespective of their taxonomically correct use of genus and species names.

2. Regeneration

Historically speaking, emphasis was on regeneration until about 1990. Following the discovery that chrysanthemums are prone to infection by Agrobacterium, allowing for its potential genetic transformation, chrysanthemum regeneration studies have been part and parcel (but de-emphasized) of transformation studies until the present (Table 1). With the knowledge that chrysanthemum transformation was fraught with shortcomings, including genotype-dependence and low transformation efficiencies, regeneration has been re- appraised in the past 2–3 years.

2.1. Establishment of in vitro aseptic cultures

Initial virus elimination of initial culture explants can be achieved by dissection of apical meristems followed by, if necessary, sterilization procedures using chemical or physical (heat) treatments (Rout and Das, 1997). Tospoviruses and viroids infect various ornamental flowers of commercial value, including chrysanthemum, resulting in commercial losses (Daughtrey et al., 1997; Chung et al., 2001).

2.2. Manipulation of morphogenesis

The ability to regenerate whole plants from tissue culture is a prerequisite for most transformation systems (Fig. 1), and has been achieved in D. grandiflora by a number of

Fig. 1. Flow diagram indicating the origin of tTCLs, and the induction of roots with the use of 10% (v/v) coconut water, 4 mg/l 2,4-D, 0.1 mg/l NAA, 1 mg/l IBA or 1 mg/l IAA; callus with the use of 0.1 mg/l BA and 0.5 mg/l NAA; shoots with the use of 2 mg/l BA and 0.5 mg/l NAA or somatic embryos by the use of 0.1 mg/l 2,4-D or 1 mg/l IAA. Following the harvest of shoots, the most important organ for chrysanthemum regeneration and transformation studies (on average 1.37 shoots per tTCL), these may be rooted in vitro on 3 g/l Hyponex- supplemented agar medium, acclimatized in the greenhouse to 100%, then induced to flower under short day conditions with a 4-h night break. J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 727 728 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 groups using various species and cultivars, basal media, different plant growth regulator (PGR) and media additive combinations and concentrations, derived organogenesis from a number of explant sources including: stems (node and internode), axillary buds, leaves, shoot tips or apical meristems, protoplasts, roots, pedicels and florets (Table 1). On stem explants, shoots originate from cortical cells, but from individual epidermal cells in flower pedicels. Indirect regeneration via callus (Fig. 2C) was reported from stem, petal and shoot tips, but adventitious shoot regeneration derived from an initial callus phase may result in somaclonal variation and in chimerism while direct shoot regeneration from leaf or stem explants may eliminate such undesirables. In petal-derived tissue culture of C. morifolium, solid mutants could be isolated, despite somaclonal variation occurring in the vegetative tissues (Pillai and Zulkifli, 2000). Generally stem material results in a greater shoot regeneration capacity than leaf (Gao et al., 2001). Chrysanthemum is traditionally micropropagated on agar solid-based, sugar-supplemented media, but has also been propagated photoautotrophically or photomixotrophically under CO2 enrichment, with reduced contamination levels and production costs (Mitra et al., 1998), and tissue-cultured plants perform just as well as hydroponic systems (Hahn et al., 1998). Despite the regeneration of whole plants (primarily adventitious shoots without an intermittent callus phase and a prerequisite for any transformation system; Fig. 2F) from the tissue culture of various explant sources (Table 1), few histologically confirmed reports on somatic embryogenesis in Dendranthema exist (May and Trigiano, 1991; Pavingerova´ et al., 1994; Urban et al., 1994; Teixeira da Silva, 2003c). A mixed organogenic response (i.e. shoots, roots, callus and embryos forming together) or multiple-organ formation (Rout and Das, 1997) is as a result of the cellular heterogeneity present in the initial explant source (Tanaka et al., 2000) but this can be overcome by the use of thin cell layers (TCLs) in conjunction with a single PGR application (Nhut et al., 2003a,b; Teixeira da Silva, 2003a,c; Fig. 1). It is believed that adventitious shoot regeneration derived from an initial callus phase may result in somaclonal variation and in chimerism while direct shoot regeneration from leaf or stem explants may eliminate such undesirables (Kaul et al., 1990), although shoot regeneration capacity in various Dendranthema cultivars can be unrelated (de Jong et al., 1993). In most studies on chrysanthemum, the shoot regeneration capacity is reported as the number of surviving explants or as the number of shoots formed per explant, and in transformation experiments, this is normally on a selective (antibiotic- containing) medium.

2.3. Root induction in vitro

Chrysanthemums are usually propagated as vegetative cuttings by dipping cut stems into formulated auxin-containing powder for adventitious root induction, the rooted plantlets being resilient to dessication, resulting in ca. 100% acclimatisation (Nishio and Fukuda, 1998). Cellulose plug-saturated liquid medium is one way of increasing survival and rooting of in vitro developed plantlets once transferred ex vitro (Roberts and Smith, 1990). Following acclimatization, a number of factors affect rooting in the greenhouse, and these are detailed elsewhere (Rout and Das, 1997). Rooting can occur independently of the presence of lateral organs and juvenility. In vitro adventitious roots can be established easily by placing in vitro-formed adventitious shoots onto a PGR-free medium, albeit with a lower J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 729

Fig. 2. Tissue culture, organogenesis in vitro and synthetic seed production. (A) Control spray-type D. grandiflora cv. ‘Lineker’ (blush pink). (B) Change in flower colour to soft pink following UV light exposure of stem explants. Culture of tTCLs results in: hard, yellow callus derived after induction with TDZ (C); somatic embryo (torpedo stage) after induction with 2,4-D (D); adventitious root induction with coconut water (E); adventitious shoot induction with NAA and BA (F). (G) No flowering differences observed in standard-type ‘Shuhou-no-chikara’ following treatment with different carbon sources, polyamines or filter paper (top left to right, respectively; see text for details). (H) Synthetic ‘‘seed’’ using encapsulated stem explants (capsule width = 4–5 mm). Bars: 1 cm = 2 mm (C), 220 Am (D). than ideal acclimatisation percentage. The in vitro induction of shoots from both stem and leaf explants has been well documented. Despite adventitious root formation (or rhizogenesis) of shoots being well documented in chrysanthemum, there is only one report on the de novo formation of roots (Teixeira da Silva, 2003a; Figs. 1 and 2E), which may allow this 730 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 731 procedure to be used for cryopreservation, artificial seed production (root ‘‘synseeds’’), secondary metabolite production (root-specific) and improvement of the genus through genetic engineering. Root development (Yi et al., 2003) or callus induction (Yiyao et al., 2002) was enhanced by sound wave treatment while electrostimulation at low field intensity (10 kV/min) resulted in an increase in the number of roots, while higher field intensity (40 kV/min) resulted in a higher average number and total length (Panfilova and Andrianov, 1996). The use of nitrate or urea was sufficient to ensure the induction and formation of roots of in vitro C. morifolium axillary shoots (de Argollo et al., 1998).

2.4. Somatic embryogenesis

The reports of somatic embryogenesis in chrysanthemum are few, and have been developed in only select D. grandiflora cultivars (May and Trigiano, 1991; Pavingerova´ et al., 1994; Urban et al., 1994; Teixeira da Silva, 2003c; Fig. 2D). The single-cell origin of somatic embryos eliminates the possibility of regeneration of chimeric plants consisting of both transgenic and non-transgenic tissues. Loss of marker genes in regeneration was through direct organogenesis—as opposed to somatic embryogenesis—have been reported (Pavingerova´ et al., 1994). Embryoids were shown to form directly on the cut edges of garland chrysanthemum leaf explants on medium containing 0.1 mg/l BA and 1 mg/l NAA, and originated from epidermal and sub-epidermal layers, and not from callus (Oka et al., 1999). Neither the choice of carbohydrate source or concentration had an effect on somatic embryogenesis, although somatic embryogenic cultures developed better in the dark (Teixeira da Silva et al., 2003, Fig. 1).

2.5. Effect of additives and other factors on morphogenesis

Numerous studies have recently been completed on the effect that a number of factors and media additives have on chrysanthemum TCL morphogenesis. To further enhance the medium-dependence of explants, TCLs were used in the experiments. TCLs, derived from cells, tissues or organs, are of a small size, excised either (a) longitudinally (lTCL), being thus composed of a few tissue types or (b) transversally (tTCL), thus composed of several tissue types, but which are normally too small to separate, as in the case of chrysanthemum. In the TCL system, the morphogenic and developmental pathways of specific organs may be clearly directed and controlled (Le and Nhut, 2000; Nhut et al., 2003a,b, Fig. 1). The effect that a number of media additives have on chrysanthemum morphogenesis, without necessarily eliminating differences to the genotype or mutations, can be eliminated by modification of the culture (Fig. 3).InRout and Das (1997) there

Fig. 3. Generalized scheme of acclimatization of chrysanthemum plants from ex vitro to in vitro and back to ex vitro. Greenhouse mother or stock plants, in which flowering occur, are maintained under short day conditions. Explants derived from mother plants are surface-sterilized, then placed on suitable organogenic medium as described in Fig. 1. Plants exposed to a gene introduction method (GIM) or stress treatment are similarly regenerated on the same medium. Hyperhydric or deformed shoots can be recovered by consecutive sub-culturing cycles (maximum 3) and with slight medium modifications. Both normal and genetically modified (GM) shoots can be similarly acclimatized to the greenhouse. 732 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 is some detailed discussion on the merits/demerits of different PGRs on chrysanthemum tissue culture.

2.5.1. Filter paper Despite the wide use of filter paper in tissue culture, only one report truly addressed its direct effect on plant tissue culture and in vitro growth and morphogenesis (Teixeira da Silva, 2003b). In that study the effect of filter paper on in vitro growth and morphogenesis of chrysanthemum tTCLs was examined, focusing on the buffering effect it has on the phytotoxicity of antibiotics; that study further explored the effect that filter paper had on chrysanthemum in vitro growth and morphogenesis (callus, shoot, root or somatic embryo formation) when placed on specific morphological programme media. Whatman #1 and Advantec filter paper positively stimulated organogenesis and buffered the phytotoxic activity of aminoglycoside antibiotics (AAs) but Whatman #3 filter paper negatively impacted chrysanthemum organogenesis.

2.5.2. Carbon sources Various carbon sources, selective agents for positive selection systems, were tested on chrysanthemum stem explants (Fukai, 1986) or stem TCL morphogenesis (Teixeira da Silva, 2003e). The rationale behind those experiments lay in the fact that the plant cannot use or metabolise all carbon sources effectively, and thus those could be used as limiting factors to regeneration, growth and development. Sucrose, glucose and fructose were shown to support metabolic growth in ‘Lineker’ and ‘Shuhou-no-chikara’ (Teixeira da Silva, 2003e), but arabinose, xylose, lactose, galactose and rhamnose could not in ‘Shuhou-no- chikara’ (Fukai, 1986). Using this principle it was possible to establish threshold survival levels (TSLs; the level at which a TCL does not differentiate independent of the increase in carbon source) in response to varying concentrations of different carbon sources, since when using a non-antibiotic marker gene, a low level of nutrient medium is utilized, making the TCL highly dependent (more heterotrophic) on the carbon source. Since the shoot regeneration capacity of chrysanthemum is severely hampered by the presence of antibiotics in the selective caulogenic medium (Teixeira da Silva and Fukai, 2001), which, despite optimisation of shoot production in the caulogenic programme, is disturbed at higher antibiotic concentrations, the search for alternative (positive) selection systems using other carbon sources may benefit the outcome and efficiency of chrysanthemum genetic transformation. TSL levels could be achieved in 64% of the carbon sources tested, enhancing their potential, provided that genes coding for their respective degrading enzymes can be cloned into a vector system. C. morifolium showed reduced shoot and root formation with an increase in concentration of osmotic agents mannitol, sucrose and sorbitol (Shibli et al., 1992). In separate studies, chrysanthemum could be better multiplied when in a photoauto- or photomixotrophic culture, supplemented only with 2% CO2 (Mitra et al., 1998).

2.5.3. Polyamines Polyamines (PAs) have been recognized as a new class of natural PGR (Tiburcio et al., 1993) and have been implicated in the control of the cell division cycle, growth, differentiation, developmental processes such as flowering, and adventitious rooting (Kaur-Sawhney and Applewhite, 1993; Teixeira da Silva, 2002a). Putrescine, spermine, J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 733 spermidine and cadaverine had both a growth-supporting and growth-inhibiting activity in different in vitro morphogenic programmes (callogenic, caulogenic, rhizogenic, somatic embryogenic; Teixeira da Silva, 2002a). Even though all PAs detrimentally affected caulogenesis and somatic embryogenesis, shoots that were harvested from the former were not significantly different from controls, not showing altered morphology and flowering when greenhouse-acclimatized. The use of PAs (spermine, spermidine and putrescine), however, positively stimulated rhizogenesis, while cadaverine reduced the rhizogenic response. Polyamine conjugates have been implicated in the regulation of flower initiation in chrysanthemum (Aribaud and Martin, 1994). In chrysanthemum in vitro leaf cultures, polyamines are covalently linked to protein substances and this affects root and shoot bud differentiation (Aribaud et al., 1995). Callus cultures are characterized by high levels of free and particularly conjugated PAs (Aribaud et al., 1999). Ornithine decarboxylase-mediated biosyntheis and diamine oxidase-mediated catabolism of putrescine occurs in the induction of in vitro rooting in chrysanthemum (Martin et al., 1997).

2.5.4. Aminoglycoside antibiotics AAs are still the most common selective agents in genetic transformation experiments both in chrysanthemum and almost every other plant species, the most common being the use of kanamycin A, gentamycin or G-418 as a selective agent for transgenic plants harbouring the nptII gene (Teixeira da Silva, 2002b; Teixeira da Silva et al., 2003). Most AAs had a negative effect on in vitro growth and morphogenesis (shoot and root formation) of chrysanthemum TCLs. The effect of the AA concentration on plant morphogenesis and explant survival depended on the size of the explant, the choice of explant source, the timing of infection by A. tumefaciens and selection pressure in genetic transformation. In separate experiments on the effect of other antibiotics on TSL values, a gradient of phytotoxicity was shown: bialaphos>chloramphenicol>rifampicin>streptomycin>minomy- cin>ampicillin>penicillin G = penicillin V (Teixeira da Silva et al., 2003). Another study (Teixeira da Silva and Fukai, 2001) showed the importance that Agrobacterium selective agent (carbenicillin, cefotaxime or vancomycin) has on maximizing Dendranthema SRC, while minimizing phytotoxicity and explant mortality. In the case of cefotaxime, shoot regeneration capacity increased (but not significantly) when added at 250 mg/l.

2.5.5. Wounding Mechanical wounding—induced by brushing leaf explant surfaces—was shown to increase the shoot regeneration capacity in D. grandiflora ‘Parliament’ (de Jong et al., 1993), while sonication in ‘Shuhou-no-chikara’ and ‘Lineker’, standard and spray-type chrysanthemums, also had a shoot regeneration stimulating effect (Teixeira da Silva and Fukai, 2003).

2.5.6. Gelling agent Bhattacharya et al. (1994) found that sago and isubgol, as gelling agents of herbal origin, and filter paper, nylon cloth, polystyrene foam or glass wool as support matrices in liquid medium could be used as effectively as agar in D. grandiflora micropropagation, or superior to it due to their reduced cost. Bu and Chen (1988) found that activated charcoal annulled the effects of 12 PGRs when added to the medium at or above 1% (w/v). 734 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766

3. Conventional breeding

Nature has played a role in inducing polyploidy in Chrysanthemum through evolution, giving rise to tetra-, hexa-, octa- and decaploids, all of which give breeders a greater understanding of the origin of the cultivars in question. Humans too have contributed, through artificial interference, to changes in chrysanthemums. Many techniques are still employed by chrysanthemum breeders to improve varieties: chromosome-doubled plants, produced by colchicine treatment, were used to produce breeding parents with improved pollen fertility (Endo and Inada, 1997), while para-fluorophenylaneline was used to successfully produce chromosome-reduced plants (Endo et al., 1994). Confirmation of hybrids and of ploidy (aneuploidy and euploidy) levels continues to be achieved by the use of chromosome counts (Endo, 1990; Aoyama et al., 1997). Correlations could be drawn between chromosome numbers and flowering period and flower number among edible chrysanthemums (Endo and Inada, 1990). The use of GISH (genomic in situ hybridization) was used to confirm the successful intergeneric hybrid between D. lavandulifolia and Ajania remotipinna (El-Twab et al., 1999) and the use of fluorescence in situ hybridization and GISH between Leucanthemella and Nipponanthemum (Ogura and Kondo, 1998; El- Twab and Kondo, 2001). Traditional and mutation breeding continue to be important in introducing new traits (Broertjies et al., 1976) such as modified photoperiod and temperature sensitivities (Fukai et al., 2000b) into the modern chrysanthemum from wild chrysanthemums (Douzono and Ikeda, 1998; Fukai et al., 2000a). Mutagenesis by chemical or physical (such as irradiation) means has been utilized in chrysanthemum to induce new genetic variation (Broertjies and Lock, 1985; Huitema et al., 1989; Preil et al., 1991; Rout and Das, 1997; Mandal et al., 2000b; Ahloowalia and Maluszynski, 2001), but there exists always the uncertainty of somaclonal variation. Genetic transformation has advantages over sexual recombination since it is the only method available in chrysanthemum to introduce resistance into existing genotypes, without further altering the genotype, provided mutations can be avoided in the transformation process. Inheritance of flower colour, especially pertaining to anthocyanin pigmentation, is analyzed by spectrophotomewtry, while genetic analysis allows for an interpretation of zygosity (Hattori, 1992b). In cases where hybridizations yield seeds that abort, embryo rescue techniques have been developed for select genera (Table 1). cpDNA PCR-restriction fragment length polymorphisms (RFLP) has been used to differentiate groups of wild Dendranthema populations into different groups (Fukai et al., 2002; Fig. 5N,O), nrDNA internal transcriber spacer (ITS) and cpDNA trnL/trnF inter- genic spacers to analyze the phylogeny of the Anthemideae (Oberprieler and Vogt, 2000; Oberprieler, 2002), while randomly amplified polymorphic DNAs (RAPDs) are used to analyze sports (cultivars derived vegetatively from a successful cultivar but differ from it in some characteristics) and chimeric plants (Wolff, 1996). RAPDs were also used to establish the genetic relationship of C. zawadskii to other related Korean wild chrysanthemums (Lee and Kim, 2000) and to study the origin of Chinese cultivated chrysanthemum (Dai et al., 1998). In cases where DNA Amplification Fingerprinting (Scott et al., 1996) cannot be used to differentiate cultivars, identify somatic mutants or radiation- induced sports, arbitrary signatures from amplification profiles, inter simple sequence repeats, PCR, hybridization-based DNA fingerprinting or RFLPs can, having application J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 735 in marker assisted breeding and in protection of plant breeders’ rights of varieties or cultivars (Wolff et al., 1995; Trigiano et al., 1998). ITSs of nuclear ribosomal DNA were sequenced, and morphological cladistic analyses, cytology and isozyme analysis were conducted to differentiate 52 species from 32 genera and eight subtribes of the Anthemideae (Francisco-Ortega et al., 1997). Chromosome studies still continue to be important in separating chrysanthemum species (Kondo et al., 1998, 1999; Suzuki et al., 2001) while, due to high ploidy, isozymes/allozymes [aspartate amino transferase (AAT), acid phosphatase, endopeptidase, esterase, glucose-6-phosphate dehydrogenase, glutamate–oxaloacetate transaminase (GOT), leucine amino peptidase (LAP), multiple dehydrogenase, peroxidase, phospho- gluco isomerase (PGI), shikimate dehydrogenase (SDH), superoxide dismutase] are effective in differentiating cultivars (Fiebich and Hennig, 1992; Roxas et al., 1993).In the case of the Chrysantheminae, the geographic origin of genera and species within it could be desciphered by the use of PAGE for AAT, glutamate dehydrogenase, isocitrate dehydrogenase [NADP] (IDH), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (PGD), SDH and triose-phosphate isomerase (Francisco-Ortega et al., 1995). Allele frequency data for polymorphic loci could be obtained when GOT-1, IDH-1/ 2, LAP-2, MDH-3, PGD-1, PGI-2, phosphoglucomutase and shikimate 5-dehydrogenase were used to differentiate different populations of Achillea (Purdy and Bayer, 1996). SDS- PAGE protein profiles may also prove as a useful marker (Williams et al., 1995).

4. Genetic transformation

The ability to transform economically important cut flower varieties would allow the use of molecular genetic techniques to modify characteristics such as flower colour, shape, height and growth morphology, longevity, horticultural traits, insect and disease resistance, and resistance to environmental stresses. Transformation has been reported in numerous groups (Tables 2–4), but low transformation efficiencies and high levels of cultivar specificity reduced the utility of these systems (Tables 2 and 3). Transformation protocols that result in a reduced transgene expression are limited, especially in engineering virus resistance where transgene expression levels are an important concern (Chasan, 1994). Traditionally new traits, variation and improved qualities are achieved through conventional breeding, while the commercial cultivars are usually propagated vegetatively through cuttings and suckers, and mass multiplication in vitro is achieved through meristem (axillary and terminal buds) or callus culture, ovule culture, protoplast culture and even somatic embryogenesis (Table 1). Even so, regeneration methods are severely hampered by high ploidy levels, and by limits of the gene pool of this aneuploid hexaploid complex [2n =54F 7–9] (Roxas et al., 1995b). Furthermore, seed production is frequently low due to incompatibility of many crosses (Boase et al., 1997), while polygenic sexual crosses can disrupt the delicate balance of uniform growth and synchronous flowering that makes specific cultivars desirable (Sherman et al., 1998b). At present, several transgenic chrysanthemum plants containing genes of interest have been successfully obtained (Tables 2–4): tomato spotted wilt virus (TSWV) resistance (Urban et al., 1994; Yepes et al., 1995); modified flower colour by chalcone synthase 736 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766

Table 2 Antibiotics (aminoglycoside and Agrobacterium-eliminating) used in chrysanthemum genetic transformation studies Principal cultivar(s) + Source AA Selection Initial Regenerationz Reference others Selectionz 1610, Parliament + 8 Leaf None None – – de Jong et al., 1990 Moneymaker Pedicel K Early 25 25 Lemieux et al., 1990 Parliament + 13 Leaf K Early 50 50–100 van Wordragen et al., 1991 Korean* Leaf, K Early 25 10 ! 20 Ledger et al., 1991 stem Peach Margaret + 4 Leaf, K Early 25 10 ! 20 Ibid. stem Parliament Leaf K Early 50 50–100 van Wordragen et al., 1992a4 1610, Parliament + 6 Leaf None None – – van Wordragen et al., 1992b Moneymaker Leaf K Late 100 100 Courtney-Gutterson et al., 1993 1610, Parliament + 6 Leaf None None – – de Jong et al., 1993 Carillon + 5 Leaf, KH Late K25 H5 K50 H10 Renou et al., 1993 stem Super White Stem K,B Early K15–25 15–25 Lowe et al., 1993 B0.5–1 B0.5–1 Parliament Leaf K Early 50 50–100 van Wordragen et al., 1993 Moneymaker Leaf K Early 100 0 Courtney-Gutterson et al., 1994 White Snowdon + 3 Leaf K Late 100 50 Pavingerova´ et al., 1994 8382, 89100, 89124 Flower K Early 10–25 0 de Jong et al., 1994 Hekla, Iridon, Polaris Leaf K Early 50 10 Urban et al., 1994 White Snowdon Stem K Early 100 100 Benetka and Pavingerova´, 1995 1581 Pedicel K Early 10 10 de Jong et al., 1995 1581 + 9 Stem K Early 10 0 Fukai et al., 1995 Bornholm, White Hurricane Leaf K Early 25 50 Dolgov et al., 1995 Iridon, Polaris + 4 Leaf, K Late 10–75 20 Yepes et al., 1995§ stem C. coronarium (garland) Root, K Early K50 K50 Oka et al., 1996 leaf Parliament + 4 Leaf KH Early K10–50 K10–50 Dolgov et al., 1997 H10–15 H10–15 Peach Margaret Leaf K Early 25 20 Boase et al., 1998a Peach Margaret + 2 + 1y Leaf KS Early K25 K25 Boase et al., 1998b 001 Leaf, K Early 50 25 ! 10 Fu et al., 1998 stem Fashion Yellow, Leaf K Early 20 20 Kim et al., 1998a Golden Glory Hekla, Iridon, Polaris Stem K Early 50 50 Kim et al., 1998b Hekla, Iridon, Polaris Leaf P Early 50 50 Sherman et al., 1998b (continued on next page) J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 737

Table 2 (continued) Principal cultivar(s) + Source AA Selection Initial Regenerationz Reference others Selectionz Shuhou-no-chikara Stem KHGe Early 15 7.5–10 Shinoyama et al., 1997 Shuhou-no-chikara Stem KHGe Early 15 7.5–10 Takatsu et al., 1998 Fashion Yellow, Leaf K Early 20 20 Young et al., 1998 Golden Glory C. morifolium Stem K Early 25 25 Shao et al., 1999 Yamabiko Stem K Early 50 10 Takatsu et al., 1999 Polaris, Golden Polaris, Leaf, K Early 5/10 25/30 Yepes et al., 1999§ Iridon stem Shuhou-no-chikara Leaf G Early 20–30 20–30 Shinoyama et al., 2000 Kanseisetsu Stem H Early 10–40 20 Shirasawa et al., 2000 Hybridy Stem K Early 100 100 Tosca et al., 2000 1581 Stem K Early 25 25 Annadana et al., 2001 Iridon Leaf, K Late 50 50 Zheng et al., 2001 stem Regan Leaf K Early 12.5 25 Ishida et al., 2002 Cheonsu Leaf K Early 50 50 Jeong et al., 2002 Shuhou-no-chikara + 3 Leaf K Late 50 50 Kudo et al., 2002 Shuhou-no-chikara Leaf G Early 20 20 Shinoyama et al., 2002 Shuhou-no-chikara, Stem K Early 30 30–50 Teixeira da Silva and Lineker Fukai, 2002a,b Regan Leaf K Early 12.5 25 Toguri et al., 2003 1581 Pedicel K Early 10 25 Petty et al., 2003 z =mg lÀ 1.*=D. indicum; *1 = artemisinin production. y = D. zawadskii Â D. Â grandiflorum. GIM = Gene introduction method (all Agrobacterium tumefaciens, except for § = Biolistics; 4 = A. rhizogenes); AAs: G = G418, Ge = geneticin, H = hygromycin, K = kanamycin, P = paramomycin, R = rifampicin, B = Basta. Selection: early (0–3 days), late (>3 days); n.s. = not specified. sense and anti-sense constructs (Courtney-Gutterson et al., 1994); resistance against the beet army worm (CPRO-DLO, 1997); transgenic plants expressing Bacillus thuringensis (bt) toxin, rolC (from A. rhizogenes), Antirrhinum majus chalcone synthase and winter flounder antifreeze protein sequence genes (Dolgov et al., 1997), resistance to gray mould (Botrytis cinerea) conferred by a rice chitinase gene (Takatsu et al., 1998), modified growth form by introduction of the tobacco phytochrome b1 gene (Zheng et al., 2001) or a reduction in plant height by the constitutive expression of the Arabidopsis gai gene (Petty et al., 2003) or the rice OsMADS1 gene (Jeong et al., 2002). Only few studies exist on virus and viroid resistance-transformed chrysanthemum (Ishida et al., 2002; Toguri et al., 2003).

4.1. Agrobacterium-mediated transformation

Susceptibility of chrysanthemum to Agrobacterium has been demonstrated as far back as 1975 (Miller, 1975), with the discovery of crown gall in different plant parts of C. morifolium due to B6 Agroinfection. Chrysanthemum-specific A. tumefaciens strains have also been isolated from D. grandiflora crown gall (Chry and CNI strains; Bush and Pueppke, 1991a; Vaudequin-Dransart et al., 1995; Ogawa et al., 2000). Early experiments conducted on 11 chrysanthemum cultivars using the leaf-disk method (Horsch et al., 1985) 738 Table 3 Studies involving transformation (primarilyAgrobacterium-mediated) of Chrysanthemum-complex members Principal cultivar(s) + Source Strain CCP L/D No. O/E OD (k) Antibiotic Concentrationz Reference others (days) 1610, Parliament + 8 Leaf LBA4404 8 n.s. 4.3–13.4 1.5 (550) CF; VA 250; 400 de Jong et al., 1990

Moneymaker Pedicel LBA4404 4 L/D n.s. 0.87 (550) VA 100–300 Lemieux et al., 1990 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. Korean* Leaf, stem LBA4404, A2002 2 L/D >3 n.s. TI 500 Ledger et al., 1991 Peach Margaret + 4 Leaf, stem LBA4404,A2002 2 L/D 0–2 n.s. TI 500 Ibid. Parliament + 13 Leaf LBA4404,A281, n.s. L/D n.s. 0.5 (n.s.) CF; VA 250; 400 van Wordragen et al., 1991 Ach5,C58 Parliament Leaf LBA4404,LBA9402 n.s. L/D 0–25R 0.5 (n.s.) CF; VA 125; 200 van Wordragen et al., 1992a 1610, Parliament + 6 Leaf LBA4404,A281,Ach5 2 n.s. 0–100% 0.5 (n.s.) CF; VA 250; 400 van Wordragen et al., 1992b Moneymaker Leaf LBA4404 4 n.s. n.s. n.s. CA 500 Courtney-Gutterson et al., 1993 1610, Parliament + 6 Leaf LBA4404 2 n.s. 1.4–4.6 0.5 (n.s.) CF; VA 250; 400 de Jong et al., 1993 Carillon + 5 Leaf, stem EHA101,Ach5,C58,Bo542 < 1 n.s. 0–90% 0.7 (660) CF 500 Renou et al., 1993 Super White Stem LBA4404,C58 + 2 L/D 0–63% n.s. CF 500 Lowe et al., 1993 5 wild + 14 Parliament Leaf A281 n.s. L/D 0–25R 0.5 (n.s.) CF; VA 125; 200 van Wordragen et al., 1993 Moneymaker Leaf LBA4404 3–5 n.s. n.s. n.s. CA 500 Courtney-Gutterson et al., 1994 White Snowdon + 3 Leaf B6S3 1 L/D 38–47% n.s. CF; TI 200; 500 Pavingerova´ et al., 1994 8382, 89100, 89124 Flower LBA4404,AGL0 2 L/D 0.03–4.3 0.4–0.8 CF; VA 250 ! 125; de Jong et al., 1994 (550) 400 ! 200 Hekla, Iridon, Polaris Leaf EHA105,Ach5,A281,Chry5 3–5 L/D 13%R 2.2 (600) CA 500 Urban et al., 1994 White Snowdon Leaf B6S3 1 L/D 38–47% n.s. CF; TI 200; 500 Benetka and Pavingerova´, 1995 1581 Pedicel AGL0 2 L/D 3–12 0.5 (540) CF; VA 250 ! 125; de Jong et al., 1995 400 ! 200 Bornholm, Leaf C58,A281 2 D n.s. n.s. CF 500 Dolgov et al., 1995 White Hurricane 1581 + 9 Stem AGL0 2 L/D 11.5 0.7–1 (540) CF; VA 250 ! 125; Fukai et al., 1995 400 ! 200 Iridon, Polaris + 4 Stem AGL0 2 L/D 11.5 0.7–1 (540) CF; VA 250 ! 125; Yepes et al., 1995§ 400 ! 200 C.coronarium Root,leaf LBA4404 2 L/D < 82% n.s. CF 100 Oka et al., 1996 Parliament + 4 Leaf A281,GV3101,C58,CBE21 3 L/D 0–3% 0.6–0.9 (600) CF 500 ! 250 Dolgov et al., 1997 Peach Margaret Leaf LBA4404 4 L/D 3.4% n.s. TI 500 Boase et al., 1998a Peach Margaret + 2 + 1y Leaf LBA4404,EHA105 + 2xMOG 4 L/D n.s. n.s. TI 500 Boase et al., 1998b 001 Leaf, stem LBA4404 4 L/D 34–85 0.5 (600) CF 500 ! 100 ! 0 Fu et al., 1998 D. grandiflora Leaf LBA4404 2 L/D n.s. n.s. CF 250 Kim et al., 1998a

y Stem LBA4404 n.s. L/D n.s. n.s. n.s. n.s. Kim et al., 1998b 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. Polaris Leaf EHA105 5 L/D 0.5–4.1 2.2 (600) CA 500 Sherman et al., 1998a Hekla, Iridon, Polaris Leaf EHA105 5 L/D 0.5–4.1 2.2 (600) CA 500 Sherman et al., 1998b Shuhou-no-chikara Leaf EHA101 3 L/D 0–100% 0.2 (600) CF 250 ! 100 Shinoyama et al., 1997, 2000 Shuhou-no-chikara Stem LBA4404 3 L/D 13–31 n.s. CF 250 Takatsu et al., 1998 Fashion Yellow, Leaf LBA4404 2 L/D n.s. n.s. CF 250 Young et al., 1998 Golden Glory C. morifolium Stem LBA4404 2 L/D n.s. n.s. n.s. n.s. Shao et al., 1999 Yamabiko Stem C58,MP90 2 L/D 6.4% 0.5 (n.s.) CF 250 Takatsu et al., 1999 Polaris, Golden Polaris, Leaf, stem LBA4404,C58,EHA105 1 n.s. n.s. 0.02–5 (600) CA; CF 1000; 500 Yepes et al., 1999§ Iridon Kanseisetsu Stem EHA101 3 L/D n.s. n.s. CA 500 Shirasawa et al., 2000 Hybridy Stem EHA101 2 L/D n.s. 1.8 (660) CF; VA 500 ! 250; Tosca et al., 2000 125 ! 100 1581 Stem AGL0 2 L/D n.s. 0.7–1 (540) CF; TI 0 ! 125; 100 Annadana et al., 2001 Iridon Leaf, stem EHA105 2 L/D n.s. 2 (600) CA 500 Zheng et al., 2001 Regan Leaf LBA4404,AGL0 3 D n.s. n.s. CF 250 Ishida et al., 2002 Cheonsu Leaf LBA4404 3 D 12–94 20x dilution CF 500 Jeong et al., 2002 Shuhou-no-chikara + 3 Leaf EHA101,LBA4404,AGL0 + 1 4 D n.s. n.s. TI 200 Kudo et al., 2002 Shuhou-no-chikara Leaf LBA4404 2 D n.s. n.s. CF 250 ! 100 Shinoyama et al., 2002 Shuhou-no-chikara, Stem LBA4404,AGL0 3–4 L/D 2.3–2.7 0.6 (550) CF 500 ! 250 Teixeira da Silva and Lineker Fukai, 2002a,b Regan Leaf LBA4404,AGL0 3 D 82 n.s. CF 250 Toguri et al., 2003 1581 Pedicel AGL0 n.s. n.s. High 0.7–1 (540) CF; VA 250 ! 125; Petty et al., 2003 400 ! 200 z = AgmlÀ 1 X ! Y, X = initial concentration early in selection, Y = final concentration later in selection; 4 = A. rhizogenes.*= D. indicum; y = D. zawadskii; CCP = co- culture period in the light (L) or dark (D); OD = optical density (k = wavelength of spectrophotometer); CA= carbenicillin; CF = cefotaxime; K = kanamycin A; R = rifampicin; TI = ticarcillin; VA= vancomycin; O/E = organ per explant; R = roots, S = shoots, SE = somatic embryos; n.s. = not specified. 739 740

Table 4 Details of chrysanthemum transformation studies Principal cultivar(s) + Transgene(s) Promoter TrE%* LtTEX LsTEX PCR Southern Others Change(s) Reference others

1610, Parliament + 8 GUS CaMV35S 0–26 GFP n.s. No No No n.s. de Jong et al., 1990 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. Moneymaker nptII,GUS,CHS CaMV35S 0.1–11 G n.s. Petals Yes No No Flower colour Lemieux et al., 1990 Korean* nptII,GUS CaMV35S 0.09 G GFP,BSA n.s. Yes Yes No n.s. Ledger et al., 1991 Peach Margaret + 4 nptII,GUS CaMV35S 0 P GFP,BSA None Yes Yes No n.s. Ibid. Parliament + 13 nptII,GUS Nos, TR-2V 100 G n.s. Callus Yes No ELISA n.s. van Wordragen et al., 1991 Parliament nptII,GUS,opines CaMV35S n.s. 42% GFP None No No Opines n.s. van Wordragen et al., 1992a 1610, Parliament + 6 nptII,GUS,opines CaMV35S n.s. 1–58 GFP None No No Opines n.s. van Wordragen et al., 1992b Moneymaker nptII,CHS CaMV35S 8 G/P n.s. n.s. Yes Yes No Flower colour Courtney-Gutterson et al., 1993 1610, Parliament + 6 nptII,GUS CaMV35S 0–16 G 0–27 GFP None No No No n.s. de Jong et al., 1993 Carillon + 5 nptII,HPT,GUS CaMV35S 9–35 G n.s. Galls Yes Yes No None Renou et al., 1993 Super White nptII,GUS,luc + 2 CaMV35S 0–36 G v,BSA Galls No No No n.s. Lowe et al., 1993 Parliament nptII,GUS,cry1A CaMV35S n.s. Callus None No No Bioassay n.s. van Wordragen et al., 1993 Moneymaker nptII,CHS CaMV35S 2.3–3.6 O n.s. Flower No Yes Northern n.s. Courtney-Gutterson et al., 1994 White Snowdon + 3 GUS,ocs CaMV35S 36 G 85 O n.s. Plant No Yes Opines None Pavingerova´ et al., 1994 8382, 89100, 89124 nptII,GUS CaMV35S 0–13 G 0.4–5 GFP Callus Yes Yes No n.s. de Jong et al., 1994 Hekla, Iridon, Polaris nptII,TSWV N CaMV35S 4–7 O Callus Plant RT Yes Western None Urban et al., 1994 White Snowdon GUS,ocs CaMV35S 13–27 O n.s. R1–R12 No Yes Opines Morphology,flower Benetka and Pavingerova´, 1995 Bornholm, nptII,y-TR CaMV35S 4 O Callus Plant No Yes No None Dolgov et al., 1995 White Hurricane 1581 nptII,GUS,cry1 CaMV35S 3–12 G n.s. Plant No No No n.s. Fukai et al., 1995 1581 + 9 nptII,GUS CaMV35S 6–16 G v,BSA Leaf (O) Yes No No None Fukai et al., 1995 Iridon, Polaris + 4 nptII,TSWV N CaMV35SL 8–73 O n.s. Plant Yes Yes No None Yepes et al., 1995§ Garland (C. coronarium) nptII,GUS CaMV35S < 0.001 G Callus Plant No No No Chimerism Oka et al., 1996 Parliament + 4 nptII,HPT,bt + 3 CaMV35S 0–4 G n.s. Plant Yes Yes No Flower colour Dolgov et al., 1997 Peach Margaret nptII,Lc Nos,35S 1.3–2.1 O Root K Leaf Yes Yes No None Boase et al., 1998a Peach Margaret + 2 + 1y nptII,GUS CaMV35S ~100 G Callus None No No No n.s. Boase et al., 1998b 001 nptII,NP-1 CaMV35S 0.7–1.7 O n.s. Plant Yes Yes No None Fu et al., 1998 Fashion Yellow, nptII,GUS CaMV35S 65–99% Callus Plant Yes Yes No n.s. Kim et al., 1998a Golden Glory

Hybridy nptII,F3V,5VH CaMV35S n.s. Callus Plant Yes Yes No n.s. Kim et al., 1998b 715–766 (2003) 21 Advances Biotechnology / Silva da Teixeira J.A. Polaris nptII,TSWV N CaMV35S 10 O n.s. Leaf Yes Yes ELISA n.s. Sherman et al., 1998a Hekla, Iridon, Polaris nptII,GUS CaMV35S 50–96 P n.s. Leaf No Yes No n.s. Sherman et al., 1998b Shuhou-no-chikara nptII,HPT,GUS CaMV35S 3.4 PO Callus Leaf Yes Yes No None Shinoyama et al., 1997, 2000 Shuhou-no-chikara nptII CaMV35S < 2.5 P Callus Plant Yes Yes No n.s. Takatsu et al., 1998 Fashion Yellow, nptII,GUS CaMV35S 77–99 G Callus Plant Yes Yes No n.s. Young et al., 1998 Golden Glory C. morifolium nptII,LFYcDNA CaMV35S n.s. n.s. Plant Yes Yes Northern Morphology Shao et al., 1999 Yamabiko nptII,RCC2 CaMV35S 0.8 O n.s. Leaf Yes Yes ELISA n.s. Takatsu et al., 1999 Polaris, Golden Polaris, nptII,N CaMV35S n.s. n.s. Plant Yes Yes No None Yepes et al., 1999 Iridon Kanseisetsu nptII,GUS CaMV35S < 38% n.s. Leaf Yes Yes No None Shirasawa et al., 2000 Hybridy nptII,Ac CaMV35S 7.8 P n.s. Leaf Yes Yes No n.s. Tosca et al., 2000 1581 nptII,GUS Lhc/dCaMv 74–76% n.s. Plant No No No n.s. Annadana et al., 2001 Iridon nptII,Nt-PHY-B1 CaMV35SE n.s. n.s. Leaf (Y) Yes No RNA Morphology,flower Zheng et al., 2001 Regan nptII, pac1 CaMV35SL 20 O n.s. Plant Yes Yes Western None Ishida et al., 2002 Cheonsu nptII,OsMADS1 Nos,35S 2.5–3.3 O Callus Plant Yes Yes RT-PCR n.s. Jeong et al., 2002 Shuhou-no-chikara + 3 nptII,HPT,GUS Nos,35S 2.8–14.5 G Callus Leaf Yes Yes RNA n.s. Kudo et al., 2002 Shuhou-no-chikara nptII, cry1Ab Nos,35S n.s. n.s. Plantlet No Yes Western n.s. Shinoyama et al., 2002 Shuhou-no-chikara, nptII,GUS Nos,35SEL 3–27 GP Callus Plant Yes No No None Teixeira da Silva and Lineker Fukai, 2002a,b Regan nptII,pac1 CaMV35SL 0.4–2 O Cell,callus Plant Yes Yes Western n.s. Toguri et al., 2003 1581 nptII,gai 35S;GGP 1–2 O Callus Plant No No RT-PCR Stunted growth Petty et al., 2003 TrE = transformation efficiency, either as *, No. positive shoots or explants/No. explants Â 100, either G, GUS-based, P, PCR-based or O, other reporter or Southern-based; TEX = transgene expression; LtTEX = localization of transient TEX, LsTEX = localization of stable TEX; 4 = A. rhizogenes;*=D. indicum; y = D. zawadskii; GFP = GUS focal point; BSA= blue staining area; v = venation; Leaf for PCR is either young (Y) or old (O); L = leader, E = enhancer; Lhc = potato Lhca3.St.1 promoter containing an MAR (matrix-associated region); dCaMV = doubles CaMV promoter (a duplication of the upstream enhancer sequence); GGP = gai gene promoter; n.s. = not specified. 741 742 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 revealed that transformation efficiency was cultivar-dependant, although the reason for successful transformation remained unknown. Bush and Pueppke (1991b) claimed that cultivar-specificity of chrysanthemum to Agrobacterium strains is independent of the endogenous levels of PGRs in the plant. Furthermore, wounding was shown to be essential for successful transformation (de Jong et al., 1993), probably through the activation of vir gene products, which in turn activate the gene transfer process. Various authors reported similar cultivar- and Agrobacterium strain-dependance (van Wordragen et al., 1991; Boase et al., 1997, 1998a). In experiments using A. rhizogenes infection of D. grandiflora ‘Parliament’, there was a low efficiency of gene transfer (i.e. lack of stable integration of the T-DNA in the plant genome) and transient transgene expression (van Wordragen et al., 1992b). A system for the transformation of chrysanthemum ‘Indianapolis White Giant #4’ was developed using A. tumefaciens LBA4404 harbouring binary vector pJJ3499. Transformants were regenerated on selection medium and gene insertion was confirmed using molecular (PCR) analysis. Chalcone synthase transformants were obtained, and tested for somaclonal variation (Lemieux et al., 1990). In separate experiments white- flowering florist chrysanthemums were produced using chalcone synthase sense and antisense constructs (Courtney-Gutterson et al., 1994). In separate experiments in which LBA4404—containing the nptII gene and the maize Leaf colour (Lc) cDNA—was used, the successful transformation of ‘Peach White’ was demonstrated (Boase et al., 1998a). Sonication-assisted Agrobacterium transformation (SAAT) is a method that involves subjecting plant tissue to brief periods of ultrasound in the presence of Agrobacterium. SAAT usually produces small and uniform fissures and channels throughout the tissue allowing the Agrobacterium easier access to internal tissues, especially tissue, such as meristematic tissue, which is buried under several other cell layers. The technique has been quite restricted in its application, and has been used on soybean, cowpea, white spruce, wheat and maize (Trick and Finer, 1997). Transient transgene expression and subsequent stable h-glucuronidase transgene expression levels increased in chrysanthemum when SAAT was used (Teixeira da Silva and Fukai, 2002b). This level differed depending on the plasmid construct and Agrobacterium strain (Figs. 4 and 5A–G). Regeneration of D. grandiflora ‘Parliament’ leaf explants following cocultivation with A. tumefaciens was severely reduced even when explants were placed on optimized regeneration media. This negative effect was corrected when a pre-culture period of 8d was used (de Jong et al., 1993). Similar results were obtained for ‘Lineker’ and ‘Shuhou- no-chikara’, standard and spray-type chrysanthemums (Teixeira da Silva and Fukai, 2002a). Both Agrobacterium-mediated transformation and shoot regeneration are triggered by wounding of the explant, and the degree and timing of explants are expected to be important factors in gene transfer and regeneration.

Fig. 4. Structure of plasmids (Courtesy of Kirin Breweries, Co., Ltd., Japan) used in transformation experiments. GUS-containing plasmid constructs: (A) pBI121 (in LBA4404); (B) pSKGN1 (bombardment) or pKT2 (in LBA4404); (C) pKT3 (in AGL0). Virus/viroid resistance gene-containing plasmids: (D) pKT66; (E) pKT61; (F) pKT2-Lpac1; (G) pKT-Mfpac1. Arrows indicate direction of transcription (5V ! 3V). E = Enhancer; L = leader sequence from soybean h-glucanase. J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 743 744 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766

4.2. Biolistic-mediated transformation

Transformation of chrysanthemum (Table 3) by A. tumefaciens has been reported in numerous groups, but biolistic-mediated transformation studies are few (Yepes et al., 1995; Teixeira da Silva and Fukai, 2002a,b). In one, tungsten particles were used as the microcarrier, and 89% of transgenic plants selected on kanamycin were PCR-positive, while 71% of these contained both genes (Yepes et al., 1995). In all, however, low transformation efficiencies and high levels of cultivar specificity reduced the utility of this system. Agrobacterium-mediated transformation is the most popular method to transform chrysanthemum (Table 3). The transformation efficiency in D. grandiflora was attributed to the regeneration procedure (Robinson and Firoozabady, 1993), the use of a binary vector (Ledger et al., 1991), the choice of selective agent (Renou et al., 1993) or gene introduction method (Teixeira da Silva and Fukai, 2002a). Acetosyringone, a phenolic compound and an inducer of T-DNA transfer enhanced transformation of chrysanthemum and increased transgene expression (Teixeira da Silva and Fukai, 2002a). Wounding of tissue (as induced by particle bombardment) has been shown to induce shoot formation in chrysanthemum (Hosokawa et al., 1998).

4.3. Inducing virus/viroid and fungal resistance

Western flower thrips is one of the most effective vectors for transmitting Tospoviruses (including TSWV, impatiens necrotic spot virus, and chrysanthemum stem necrosis virus Nagata and de A´vila, 2000) and other viruses (chrysanthemum mild mottle virus, chlorotic ring mosaid virus, or chrysanthemum B carlavirus) to greenhouse plants (Silva et al., 2001). Tospoviruses infect various ornamental flowers of commercial value, including chrysanthemum, petunia, impatiens and snapdragon (Daughtrey et al., 1997). Supplemen- tal blue-light in the greenhouse enhances Orius oncidiosus, a natural predator of western flower thrips, reproduction, especially in biological control programmes (Stack et al., 1998). Other diseases caused by single-stranded circular RNA, viroids (such as chrysanthemum stunt viroid, or chrysanthemum chlorotic mottle viroid), in vegetatively propagated plants cause stunting (reduction in growth rate), causing a loss of commercial value (Chung et al., 2001). Most of these diseases are caused by RNA-containing pathogens and one possible mechanism of eliminating them is by introducing a double-stranded RNA- specific ribonuclease such as pac1 enzyme from Schizosaccharomyces pombe, a yeast. Pac1 ribonuclease has been successfully used to induce virus (tobacco mosaic virus, and cucumber mosaic virus) resistance in tobacco (Watanabe et al., 1995) and potato (potato spindle tuber viroid; Sano et al., 1997) since it targets dsRNA, which occurs in plant viruses that replicate via dsRNA intermediates and in viroids, which despite having ssRNAs, these form dsRNAs during rolling-circle replication. Other researchers attempted to introduce the nucleocapsid (N) gene into chrysanthemum to provide resistance against some TSWV strains, but the N protein could not be detected by ELISA (Yepes et al., 1995; Sherman et al., 1998a,b). In the latter two studies, resistance is based on the structural properties of each virus, which can suffer changes occasionally. Transgenic D. grandiflorum ‘Regan’ containing the pac1 gene were found to be resistant to chrysanthemum stunt viroid (Ishida et al., 2002; Toguri et al., 2003). J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 745

Another form of introducing virus/viroid resistance into plants is by using the mammalian (and other higher vertebrates) 2–5A system (Mitra et al., 1996). Interferons (a and h) are produced by virus-infected cells and act as signal molecules for adjacent non-infected cells. Interferon-stimulated genes are activated, whose proteins are respon- sible for antiviral responses, such as the 2,5-A system. Two enzymes are necessary for a functional 2,5-A system: (i) 2–5-A synthetases to produce 2,5-A and (ii) 2,5-A-dependent RNaseL, which, once activated by 2,5-A, cleaves viral and cellular ssRNAs. This 2,5-A system proved successful (Mitra et al., 1996) in reducing (but not eradicating, measured by ELISA), tobacco mosaic virus, alfalfa mosaic virus, and tobacco etch virus. Chrysanthe- mum yellows phytoplasma, a plant pathogenic mollicute belonging to the 16Sr-IB genetic group which infects numerous dicotyledonous plants, including chrysanthemum, is limited to phloem infection (Palermo et al., 2001). No resistance (classical or molecular) to chrysanthemum yellows exists. In one report on the genetic transformation of chrysanthemum, transgene inactivation was reported 1 year after transgene insertion (Takatsu et al., 1999). Another chrysanthemum transformation report claimed chimerism caused by transgene inactivation, primarily by methylation (Shirasawa et al., 2000). Gene silencing has been shown to occur in transgenic plants affecting genes controlling virus resistance and flower colour (Johansen and Carrington, 2001), and its trigger may be a dsRNA (Bass, 2000). Transgene inactivation or silencing was also reported in other chrysanthemum transformation studies (Pavingerova´ et al., 1994; Benetka and Pavingerova´, 1995; Shinoyama et al., 2002). van Wordragen et al. (1992b) also claimed that GUS transgene expression started later in transgenic chrysanthemum than tobacco (5 vs. 2 days) when the CaMV35S promoter was used. Possible transgene truncations or aberrations led to smaller protein sizes in transgenic chrysanthemum expressing a Bt-toxin gene compared to pure protein controls (Shinoyama et al., 2002). Similar results were obtained in Western results of transgenic chrysanthemum plants transformed with Pac1, 2,5-A and RNaseL genes (Teixeira da Silva and Fukai, unpublished results; Fig. 5H–M). Only a single study has addressed resistance against gray mould (Botrytis cinerea) using transgenic technology. A rice chitinase gene, RCC2, was cloned into cultivar ‘Yamabiko’ using A. tumefaciens strains C58 and MP90, resulting in the production of 11 resistant lines (Takatsu et al., 1999). The potential spread of transgenic pollen to wild populations, especially from spray- type chrysanthemums, may limit progress of transgenic research and slow legalization and patent application of genetically modified plants.

4.4. Modifying growth form

The control of height and form in chrysanthemum is one of the major targets for production of small, compact pot plants. One of the most common methods of inducing dwarfing in chrysanthemums is through the use of growth retardants such as gibberellic acid (GA), whose use poses an expensive and potentially environmentally damaging solution. Recently a number of GA-insensitive genes from Arabidopsis thaliana mutants have been cloned, including spindly (spy), short internodes (shi), and GA insensitive (gai), all of which result in stunted growth (Teixeira da Silva and Nhut, 2003). Reduced height, 746 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 that is, shorter and more compact plants, and altered GA response could be achieved when the gai mutant gene, driven by its own promoter, was introduced into cultivar ‘1581’ using AGL0 Agroinfection (Petty et al., 2003). Of 29 lines expressing the gai transgene, 19 were visibly dwarfed. In a separate study, the ectopic expression of a tobacco phytochrome B1 (phy-B1) gene resulted in growth reduction, similar to that observed if a commercial growth retardant had been used (Zheng et al., 2001). The consequent inhibition of stem elongation and increase in harvest index was attributed to a hypersensitivity to far red light. Ectopic expression of

J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 747 tobacco phy-B1 delayed flowering (both induction and development), even when short-day conditions were applied. Although 32 kanamycin-resistant plants were obtained, only four independently transformed lines were identified by PCR and RNA blot analysis.

4.5. Transformation efficiencies

TrEs reported in the literature for chrysanthemum range from 0% to 35% (Rout and Das, 1997), the factors most affecting the improvement of transformation efficiencies are: genotype-dependence of regeneration, gene introduction method, timing of selection pressure, and uncertainty concerning the origin of transgenic organ formation. TCL regeneration studies were conducted in an attempt to improve the transformation efficiency. A high level of transgenic shoots and transformation efficiency can be achieved due to the small size of TCLs, despite a lower shoot regeneration capacity, when Agrobacterium (ideal vector) infection of TCLs is followed by a high (30 mg/l) selection (kanamycin) pressure. Transformation efficiencies in different Agrobacterium-mediated transformation studies of D. grandiflora range from < 0.1% for ‘Moneymaker’ to a high of 0.6% in ‘1581’ (Ledger et al., 1991; Renou et al., 1993; Courtney-Gutterson et al., 1994; Pavingerova´ et al., 1994). Transformation efficiencies could be improved from 0.1% to 0.5% in ‘Iridon’ (Urban et al., 1994). By modifying the regeneration procedure, high transformation efficiencies were obtained in ‘Hekla’ and ‘Polaris’ (Sherman et al., 1998b), with 4.1% and 1.7%, respectively, while a 2.5–4.6% transformation efficiency for four cultivars were obtained in a biolistic- mediated transformation procedure (Yepes et al., 1995). Improvement of transformation efficiencies has been attributed to a four-step plant regeneration procedure coupled with Agroinfection: callus induction, shoot primordia, shoot elongation and rooting (Sherman et al., 1998b). The transformation efficiency values presented in a study for ‘Lineker’ and ‘Shuhou-no-chikara’ fell into the range of 2–27% (GUS positive) and 0–21% (PCR positive) for bombardment related treatments, and in the range of 7–25% (GUS positive)

Fig. 5. Genetic transformation and genetic studies in chrysanthemum. Transient GUS expression in in vitro stem explant 72 h (A, GUS focal point) after particle bombardment. Agroinfection with pKT2 results in weak transient transgene expression (B) localized at cut surfaces only (72 h after Agroinfection). The use of AGL0 (pKT3) results in a greater percentage GUS transient transgene expression (C). Predominant GUS expression in venation (D) and shoot tips (E) of 6–8-week-old in vitro plantlet leaves. Transformant (F) grown on 30 mg/l kanamycin shoot-induction medium containing both gus and nptII gene constructs. PCR analysis (G) of GUS transient and stable gene expression. Lane 1: size marker; Lane 2: purified pSKGN1 from LBA4404, GUS = 954 bp, upper band, nptII= 438 bp, lower band; Lane 3: negative control, in vitro ‘Lineker’. Stable transgene expression at 1 month (Lane 4) after Agroinfection with AGL0 (pKT3). Western blot analysis (H) of Pac1 protein expression after AGL0 (MF-Pac1) Agroinfection. Lane 1: size marker; Lane 2: non-transformed ‘Shuhou-no-chikara’; Lane 3: positive Pac1 transformant ‘Regan’ (Kirin Brewery); Lanes 4–7: various Pac1 positive ‘Shuhou-no-chikara’ transformants, lane 6: a low-expression line, lane 7: a high-expression line. PCR analysis of LBA4404 (pKT61) Agroinfection trials showing: Lane 1 (size marker), lane 2 (positive control purified pKT61 plasmid), lane 3 (negative control ‘Shuhou-no-chikara’), lane 4 (positive transformant line #91 ‘Shuhou-no-chikara’) for 2,5-A (I), nptII (J), pac1 (K) and RNAase L (L). Southern blot analysis (M) of 2,5-A gene showing a 1,2 kb fragment in ‘Shuhou-no-chikara’ transformants (lanes 3–6) and a 5,7 kb fragment in one transformant (lane 6) after restriction with HindIII; lane 1 positive control (purified plasmid pKT61); lane 2 negative control ‘Shuhou-no-chikara’. cpDNA PCR-RFLP analysis of Dendranthema yoshinaganthum following restriction with XbaI (N) and StyI (O). 748 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 and 0–16% (PCR positive) for Agroinfection related treatments (Teixeira da Silva and Fukai, 2002b). PCR screening of D. grandiflora explants exposed to Agroinfection reveals that the GUS gene was expressed in 100%, 83% and 75%, respectively, of transformed ‘Polaris’, ‘Hekla’ and ‘Iridon’ plants (Sherman et al., 1998b), in which 23 and 3 PCR positive transgenics were recovered from 275 and 59 GUS positive plants in in vitro and greenhouse treatments, respectively, translating into a 8.4% and 5.1% transformation efficiency, respectively. Transformation efficiency (number of GUS positive plants per number of greenhouse-acclimatized plants) levels differed, depending on the treatment and explant source: 7% for late hygromycin selection, 14% for high initial hygromycin selection or 31% for delayed hygromycin selection when leaf or internode EHA101-Agroinfected explants were used, and 9% when nodes were used on early selection medium (Renou et al., 1993). Very high transformation efficiency values (GUS-based) were obtained in AGL0- infected D. grandiflora, ranging from 3% to 45% for 6 and 24 h, respectively (de Jong et al., 1995). Genotype-dependence led to a 0–12.5% transformation efficiency in AGL0-infected D. grandiflora transformation experiments (Fukai et al., 1995). A 4–7% and 2–3% transformation efficiency—for rooting capacity and PCR based transformation efficiency, respectively—was reported when D. grandiflora ‘Iridon’ was infected by EHA101 harbouring the pTSWV nucleocapsid gene (Urban et al., 1994), but a 0.5% transformation efficiency was observed in biolistic transformation of the same gene (Yepes et al., 1995). Of the 82 PTs, 89% had both nptII and nucleocapsid TSWV genes. A 12% transformation efficiency was observed in transgenic D. grandiflora 63 days after infection with AGL0 (de Jong et al., 1995). A 1–4% transformation efficiency was reported for 5 D. grandiflora cultivars when transformed with the bt toxin, rolC, chs and AFP genes harboured in different cointegrative and binary vector systems (Dolgov et al., 1997). A Southern-based transformation efficiency of 1.3% was obtained in transgenic D. grandiflora ‘Peach Margaret’ whose leaf explants were inoculated with LBA4404 harbouring the Lc gene (Boase et al., 1998a). A < 1% transformation efficiency was observed (ELISA or Southern-based) in transgenic spray-type chrysanthemum expressing a rice chitinase gene (Takatsu et al., 1998). Electrotransfection was shown to cause 50% mortality in chrysanthemum axillary shoots, while about 25% showed transient GUS transgene expression (Burchi et al., 1995). Over 5 min sonication in ‘Lineker’ and ‘Shuhou-no-chikara’ caused exponential explant mortality, but increased GUS transient transgene expression and blue-staining areas (Teixeira da Silva and Fukai, 2002a).

4.6. Effect of different parameters on transient GUS and transgene expression

An extended explant pre-culture period from 0 to 8 days almost tripled the observed number of blue spots in LBA4404 Agroinfected D. grandiflora ‘1610’ leaf disks (de Jong et al., 1990). In ‘Lineker’ and ‘Shuhou-no-chikara’ an increased pre-culture period positively affected explant survival in both cultivars both with particle bombardment and Agro- infection (Teixeira da Silva and Fukai, 2002a), but also an increase in escape (non-transgenic plant) formation. An early peak in GUS transgene expression has been attributed to the expression of non-integrated copies of T-DNA. This transient expression diminishes rapidly to be replaced by the much lower frequency of stable integration (de Jong et al., 1994). J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 749

Transgene expression was shown to be strain-dependent, independent of the cultivar used, similar to studies in which greatest transgene expression occurred when wild type CHRY5 and A281 were used (Urban et al., 1994). In vitro-derived leaf explants had higher transient GUS transgene expression levels than greenhouse-derived leaves, independent of the D. grandiflora cultivar and A. tumefaciens strain (Boase et al., 1998b). This could be similarly observed in both ‘Lineker’ and ‘Shuhou-no-chikara’ stem explants, but only with the application of Agroinfection, Agrolistics or SAAT (Teixeira da Silva and Fukai, 2002b). Greenhouse explants had lower transient transgene expression than their in vitro counterparts. In transformation events utilizing Agroinfection of seven D. grandiflora cultivars by wild-type Agrobacterium strains, GUS transgene expression was localized mostly near the edge of the explant, most often close to the basal cut site of a major vein, which was coincidentally the predominant site for regeneration of adventitious shoots (van Wordra- gen et al., 1992a). This would suggest that cells that are competent for regeneration have some features in common with cells that are competent for transformation. In other studies, an increased GUS transgene expression in older (basal) leaves than in younger (apical) leaves, independent of the cultivar, and the treatment (Teixeira da Silva and Fukai, 2002b). Moreover, GUS expression is primarily found in the veins and in the mid-rib, consistent with the fact that despite CaMV-35S being a constitutive promoter, that it has a strong transgene expression in the vascular tissues. The existence of blue-staining areas suggests high gene transfer efficiency, and not a cell-to-cell transport of transcription/translation products of the GUS gene, since there is the existence of single blue-stained cells (van Wordragen et al., 1992a). Chimerism, developed through mutation induction, has been achieved in C. morifolium through adventitious shoot regeneration of sectorial mutations in the floret (Chakrabarty et al., 1999). In this way, solid mutants can be isolated from mutated chimeric sectors of flower heads. GUS transgene expression chimerism, the presence of both transformed and untransformed cells within the same plant, may be one of the primary reasons to explain the spatial expression of GUS transgene expression observed in ‘Lineker’ and ‘Shuhou- no-chikara’ transformants (Teixeira da Silva and Fukai, 2002b). The level and timing of selection (in all chrysanthemum transformation studies, antibiotics are used) also affect the presence of chimerism (Table 2). A lack of systemic stable transgene expression (i.e. chimerism) may explain the lack of ELISA or Western confirmation of the TSWV nucleocapsid gene (Urban et al., 1994) and the low transgenic lc expression in ‘Peach Margaret’ (Boase et al., 1998a), and of the RCC2 gene in ‘Yamabiko’ (Takatsu et al., 1999) chrysanthemum transformation studies.

4.7. Stable transgene expression

For stable transformation, it is necessary for the T-DNA to be incorporated into the host DNA, and T-DNA that is not integrated is gradually lost and inactivated (Boase et al., 1998b). This phenomenon is evident in the GUS transgene expression in the intron- containing plasmids (pSKGN1, pKT2 and pKT3) where such a gradual loss can be seen by a decrease in the number of GUS focal points up to 72 h and a total loss of GUS focal points by a maximum of 1 week following a gene introduction method (Teixeira da Silva and 750 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766

Fukai, 2002a). The amount of Agroinfection of explants was reduced to 2% after 2 weeks of culture on selective MS medium, indicating that the GUS-positive and PCR-positive blue- staining areas were as a result of stable transformation and transgene integration, and not as a result of Agroinfection. Experiments with Agroinfection of 11 D. grandiflora cultivars with LBA4404 showed that there was no significant reduction in the number of GUS focal points over 2 weeks, indicating a possible stable integration and subsequent transgene expression of the T-DNA (de Jong et al., 1990, 1993). Large differences were observed between cultivars with some leaf explants exhibiting as little as 0 and as many as 26 GUS focal points per leaf disk when measured 6 days after infection. LBA4404 (pKIWI110) infected D. morifolium and D. indicum showed that 83% of leaf explants showed GUS activity 48 h after infection (Ledger et al., 1991). A. rhizogenes transformation experiments in D. grandiflora ‘Parliament’ showed that 42% of leaf explants expressed the GUS and iaa genes 6 days after inoculation, but 0% at 21 days, indicating the transient and non-integrated nature of the transgene expression (van Wordragen et al., 1992a). Separate experiments by the same group on seven D. grandiflora cultivars indicated that the use of A281 was far superior to Ach5, and both GUS and opines were detected at earliest 5 days after infection, indicating only the transient nature of transgene expression (van Wordragen et al., 1992b). Intron-containing plasmids (pSKGN1/pKT2 and pKT3) had an increase in transient transgene expression from 0 to 72 h (observed by the number of GUS focal points) and higher stable GUS transgene expression levels increasing from 1 week after gene introduction method ( c BSAs), the former plasmid type with fewer GUS focal points and blue-staining areas than the latter (Teixeira da Silva and Fukai, 2002a). The choice of promoter also affects the strength/level of stable GUS transgene expression (Annadana et al., 2001). Even when the commonly used CaMV promoter is enhanced by a double enhancer or by the attachment of a matrix-associated region, stable GUS transgene expression is still lower than with the use of the Lhca3.St.1 potato promoter (Annadana et al., 2001).

4.8. Localization of transgene expression

In all but one (Boase et al., 1998a) transformation study on chrysanthemum, the exact origin of the plant material used for gene analysis was not specified. In the study by Teixeira da Silva and Fukai (2002b), a higher GUS stable transgene expression observed in older leaves than in younger leaves, suggesting a greater amount of GUS protein accumulation in the former (Fig. 5D,E).InD. grandiflora (de Jong et al., 1994) similar GUS expression and plantlet–age relationships, and dominant mid-rib and vein stable transgene expression were recorded, primarily in the phloem. This vein-specific GUS stable transgene expression is characteristic of the 35S promoter (Odell et al., 1985).

5. Cryopreservation and germplasm storage

Cryopreservation is an important method (recognized by the FAO, IPGRI, IBPGR and CGIAR) for the conservation of plant genetic resources (Rout and Das, 1997; Engelmann, J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 751

2000). Freeze preservation in liquid N2 immobilizes metabolic activity, thus suspending changes that may arise in the plant cell genome. Storage of chrysanthemum genetic resources has been achieved through cryopreservation (described below), low temperature preservation (Fukai and Oe¨, 1986; Fukai, 1995; Roxas et al., 1995a; Rajapakse et al., 1996), and room temperature preservation (Hosoki, 1989). In vitro growth may be suppressed with the use of uniconazol (Fukai, 1995). Successful cryopreservation of chrysanthemum shoot tips, axillary buds or synthetic seeds (encapsulated–dehydrated explants, Figs. 2H and 6) involves the ability to regenerate thawed shoots or ‘‘synseeds’’ as well as maintaining their genetic composition. Cultivar differences were observed in the shoot regeneration capacity of 18 Japanese chrysanthemum species following cryopreservation of shoot tips in liquid N2 for 15 min in a 10% DMSO and 3% glucose solution (Fukai, 1990, 1992; Fukai and Oe¨, 1990; Fukai et al., 1991). In similar studies, axillary buds were used (Ahn, 1995). Cryopreservation is used to establish the true- to-type preservation of chrysanthemum (Fukai et al., 1994), especially in some cultivars where the apical dome has a chimeric structure, which is often disrupted in tissue culture and micropropagation (Stewart and Dermen, 1970; Bush et al., 1976). This is evident in ‘Apricot

Fig. 6. A generalized scheme for the production of ‘‘syn-seed’’ by a mixture of encapsulation–dehydration and cryopreservation (based on Sakai et al., 2000). After choosing a desirable target explant, it is pre-cultured on a sucrose-supplemented medium, encapsulated and osmoprotected using a 2% alginate solution. Following rapid cooling (vitrification) for the desired period of time, rapid warming is conducted and alginate balls are plated onto the desired regeneration medium. 752 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766

Marble’, which demonstrates a periclinal chimera, having anthocyanins in the epidermis (L1) of the apical dome and carotenoids in the mesophyll (L2) but not in L1 (Stewart and Dermen, 1970; Shibata and Kawata, 1986; Fukai et al., 1994). The use of floret or receptacle culture has been shown to eliminate the possibilities of periclinal chimerism, since shoots are derived from the epidermis/L1 (Malaure et al., 1991a; Hattori, 1992a). Encapsulation–dehydration or ‘‘synseed’’ production, a compromise between vitrification and dehydration, is an alternative for chrysanthemum cryopreservation. In this process, shoot apices were precultured in sucrose and simultaneously osmoprotected with a glycerol/sucrose mix, then dehydrated with dry silica gel prior to emerging into liquid N2 (Sakai et al., 2000; Fig. 6). Cryopreservation of C. cinerariaefolium, or Dalmatian pyrethrum, was achieved by a 3- day pre-culture period in sucrose-enriched medium, and using a 7.5% DMSO cryoprotectant, with an average cryopreservability rate at 62% (Hitmi et al., 1997, 1998a,b, 1999a). Cryopreservation, however, did not affect the biosynthetic properties, the composition or the amount of pyrethrins (Hitmi et al., 2000a). Sucrose was shown to be an effective cryoprotectant to confer freezing tolerance to pyrethrin cell cultures (Hitmi et al., 1999a,c, 2000b).

6. Postharvest biotechnology

Chrysanthemum is generally a long-lasting (in some cases 20–30 days) cut flower species, and this has been attributed to a low ethylene production during senescence (Bartoli et al., 1997a). This is due to the fact that chysanthemum is a non-climacteric species, whose flowers senesce irrespective of the presence of ethylene, often in response to changes in carbohydrate content (Adachi et al., 1999). SDS-PAGE protein profiles proved useful in identifying the stage of senescence of a flower, which could be used to predict the potential longevity of the flower at marketability (Williams et al., 1995). A reduction in cut flower quality has been attributed to the formation of air embolisms that partially or completely block the water transport from the vase solution to the rest of the cut flower stem, and as a result of increased hydraulic resistance may cause severe water stress (van Ieperen et al., 2001). Moreover, xylem occlusions induced by wounding result in physiological oxidation due to the activity of peroxidase and cathecol oxidase (van Doorn and Vaslier, 2002).GA3 and BAP were found to prolong leaf chlorophyll content while reducing leaf yellowing in D. grandiflora cvs. Seolpoong and Baekyang cut flowers (Suh and Kwack, 1994). Radiation-induced damage of gamma(a˜)-ray (Nakahara et al., 1998) or electron beam (Dohino and Tanabe, 1994) induced irradiation of cut stems, effective in the elimination of spider mite and flour beetle (Hayashi et al., 1998), could be reduced by the addition of 2% sucrose (acting as a respiratory substrate) to the vase solution (Hayashi and Dohino, 1995; Kikuchi et al., 1998). In some instances, gamma rays were shown to enhance the vase life of cut flowers (Huang and Huang, 1993). The use of sucrose, glucose, fructose or maltose was equally effective in delaying bloom wilting and foliage yellowing caused by gamma irradiation (Hayashi and Todoriki, 1996). Sucrose was shown to be an effective promoter of flower bud opening when used in pulsing solutions (Roncancio et al., 1995). Ageing of petals is accompanied by a ninefold increase in oxygen radical generation, and a J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 753 twofold increase in the content of oxidized proteins (Bartoli et al., 1997b). Tap water is superior to deionized water since the presence of calcium chloride within it delayed the increase in hydraulic resistance in the cut flower stem and decreases the transpiration rate (van Meeteren et al., 2000). In separate pulsing solution trials, various germicides were tested, as well as PGRs to maintain the green colour of leaves and turgidity, and vase-life extension was attempted by the application of inhibitors of ethylene production, with the optimal pulsing solution being distilled water + citric acid + sucrose + 8-hydroxyquinoline sulphate (HQS) or AgNO3 +GA3 (Florez et al., 1996). Methanol, ethanol, BA and paclobutrazol also retarded senescence in leaves of cut flower stems (Petridou et al., 2001). Delay of leaf yellowing and senescence processes were enhanced by chlorsulfuron, a cytokinin (Yakimova et al., 1996). A mixture of methyl bromide (Helyer and Ledieu, 1989), phosphine and CO2 caused no chemical injury to cut flowers of six chrysanthemum cultivars but was effective in the control of eight species of insect and arthropod pests (Kawakami et al., 1996).CO2 enrichment at 1000–2000 ppm increased stem length, fresh weight, number of leaves and extended vase life (Tanigawa et al., 1995). Separate studies demonstrated the effectiveness of silver thiosulphate or HQS in extending the vase life of cut flowers, while carbonated water (20–30%) was a good preservative solution for cut chrysanthemum flowers (Lee et al., 1996). The use of modified atmosphere packaging in transport of cut flowers using polyethylene and polypropylene reduced respiration rate (Yamashita et al., 1999).1- Methylcyclopropene were effective in increasing shelf-life of cut chrysanthemum flowers only when these were in the presence of other ethylene-producing fresh commodities (Able et al., 2003). Present Japanese quarantine is attempting to reduce the amount of methyl bromide used, and the present quarantine treatment for cut flowers is: 14 g/m3 methyl 3 bromide, 3 g/m hydrogen phosphide and 5% CO2 (Kawakami, 1999). A more comprehensive analysis of the processes underlying cut flower quality and postharvest maintenance can be found elsewhere (Teixeira da Silva, 2003d).

7. Other advances

Mechanization of cut flowers by a computerized sorting system has been developed with a maximum output of 2549 cut stems per hour, within defined stem length and thickness boundaries (McFarlane, 1993). In an ironic development to conventional pathogen-resistance, in vitro chrysanthemum plants were used as in planta hosts to maintain Puccinia horiana cultures over long periods (Ohishi et al., 2000; Takatsu et al., 2000) or of mycoplasm-like organisms (Bertaccini et al., 1992).

8. Conclusions and future perspectives

Advances in transgenic biotechnology of members of the Chrysanthemum-complex are in part possible due to improvements and new and significant findings in regeneration protocols. The capacity to manipulate morphogenic pathways allows for organ-specific 754 J.A. Teixeira da Silva / Biotechnology Advances 21 (2003) 715–766 control of regeneration, which in turn results in ‘‘pure’’ organogenic cultures that can be used for synthetic seed production or organ-specific secondary metabolite extraction. These organs can be successfully (cryo)stored in genebanks and germplasm storage centres by using room or low temperature strorage and cryopreservation techniques. These do not affect the final morphology or genetic stability of greenhouse plants or flowers. The longevity of cut flowers, the end-product of most Chrysanthemum-complex species, can be extended by manipulating the vase solution. Immediate goals in chrysanthemum biotechnology, some of which are presently being addressed, are: systemic virus and viroid resistance, novel flower colour and shape induction, greenhouse growth and architecture modification, bioreactor mass production of plantlets or organs, and secondary metabolite production.

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